For Peer Review10.1007...For Peer Review 1 Highly sensitive real-time PCR method to identify species...

For Peer ReviewHighly sensitive Real-Time PCR method to identify species

origin in heparinoids

Journal: Analytical and Bioanalytical Chemistry

Manuscript ID ABC-01707-2019.R1

Type of Paper: Research Paper

Date Submitted by the Author: 17-Oct-2019

Complete List of Authors: Pecorini, Simone; Biofer, Quality ControlTorrini, Lucia; Biofer, Research and DevelopmentCamurri, Giulio; Biofer, Quality ControlFerraresi, Roberta; Biofer, Quality Control

Keywords: Heparinoid, PCR, Origin, Glycogen, Purification

Analytical & Bioanalytical Chemistry

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Highly sensitive real-time PCR method to identify species origin in heparinoids

Simone Pecorini1, Giulio Camurri1, Lucia Torrini2, Roberta Ferraresi1,*,

1 Microbiology Quality Control Laboratory, Biofer S.p.A., via Canina, 2, 41036 Medolla (MO), Italy

2 Research and Development Laboratory, Biofer S.p.A., via Canina, 2, 41036 Medolla (MO), Italy

*Corresponding author, email: [email protected]

ABSTRACT

Heparinoids are the starting material for sulodexide production, drug used as intravenous anti-

coagulant, as an alternative to heparin. The origin determination in the starting material for

sulodexide, heparin and derivatives, is crucial for the safety (including the impact related to Bovine

Spongiform Encephalopathy) and efficacy of the final products. Therefore, European countries

have decided to approve the production of heparin only from porcine intestinal mucosa.

PCR (Polymerase Chain Reaction) methods are available to evaluate the origin species of crude

heparin, during heparin production process, while they lack for the same analysis in heparinoids

during sulodexide manufacturing processes.

Notably, two main critical issues occur during the origin determination by using PCR for heparinoid

analysis: first, heparin has been known to inhibit DNA polymerase activity and, second, the DNA

amounts are very low in these samples. To overcome these critical issues, our proposed method is

based on two fundamental steps, the DNA concentration by glycogen treatment and DNA

purification, which occur before and after DNA extraction, respectively. Finally, by applying Real-

Time PCR, we amplify three specific DNA sequences of ruminant species (bovine, ovine and

caprine), to assess possible contamination, and one from swine, to confirm the origin species. To

date, such method is the only one that determines origin species by PCR for heparinoids, that

guarantees quality, safety and traceability of heparin-derived pharmaceutical products. In

conclusion, our proposed method is alternative to Nuclear Magnetic Resonance and ELISA

methods, because Real-Time PCR offers significant advantages in sensitivity, specificity and

robustness.

Keywords Heparinoid, PCR, glycogen, purification, origin, glycosaminoglycans

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INTRODUCTION

After the advent of Bovine Spongiform Encephalopathy (BSE), European countries have decided

to approve the production of the heparin only from porcine intestinal mucosa [1, 2]. To ensure the

safety of these anti-coagulant agents, pharmacopoeias, institutions and monitoring organizations in

medical and pharmaceutical fields declared that the origin of the crude heparin (raw material) must

be known and certificated [2-4].

Chemically, the significant differences among porcine and ruminant (bovine, ovine and caprine)

heparins are represented by the greater abundance of sulfated groups in the first, the differences

in chemical structures, the affinity to bind the anti-thrombin III and the molecular weight distribution.

Notably, concerning anti-coagulant activity, porcine and ovine heparin are quite similar, while

bovine heparin biological activity is weaker [5].

Sulodexide is a key pharmaceutical heparin-like product widely used in thrombosis field, as a pro-

fibrinolytic agent. It is a glycosaminoglycan derived from porcine intestinal mucosa, constituted by

chains of polysaccharides, with different grades and types of sulfation sites [6]. The major

constituents of sulodexide are heparan sulfate and dermatan sulfate, which counts, respectively,

for about 80% and 20% of the total [7, 8]. While crude heparin represents the raw material for

heparin production, heparinoids (HPDs), heparin-derived glycosaminoglycans, constitute the

starting material of sulodexide manufacturing process. Heparin, differently from sulodexide and

HPD, is the only product with a dedicated monograph in European and United States

pharmacopeia.

Pharmacopeia monographs suggest adopting Polymerase Chain Reaction (PCR) as the elective

method to determine origin species. In literature, the detection of ruminant contamination has been

described with different methods, based on immunology, microscopy or spectrometry. While

microscopic and spectrometric methods lack sensitivity, ELISA is more accurate, but a matrix

interference and false positive results may occur [9, 10]. Real-Time PCR represents a valid control

method for the evaluation of the origin of heparin-derived samples, compared to Nuclear Magnetic

Resonance (NMR) [11]. Moreover, Real-Time PCR offers significant advantages in sensitivity,

specificity, accuracy and robustness [12]. PCR methods are known for origin determination in

heparin, but a gap in knowledge exists concerning the determination of origin species in HPD [1].

Generally, the main critical issue, encountered during the development of PCR method for heparin-

containing samples, is the presence of heparin, inhibitor of DNA polymerase [13-15].

The aim of our study is the identification and validation of a Real-Time PCR method to determine

origin species In HPD samples. Therefore, it will be possible to ensure the quality, safety and

accuracy controls during the manufacturing process of sulodexide, as it happens for heparin.

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https://en.wikipedia.org/wiki/Glycosaminoglycan

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MATERIALS AND METHODS

No animals were used for this study. The study did not violate human rights.

DNA assay by optical density at 260 nm

Total DNA concentration was assayed by ultraviolet (UV) absorption at 260 nm, by using the UV-

VIS spectrometry (Perkin Elmer, Waltham, USA) with three replicates. Sample with absorbance

more than 1.0 AU (Absorbance Units) was diluted 1:10 or 1:100, from an initial concentration of 4

mg of powder per mL of water. Blank measurements were performed by using molecular biology

grade water.

Nucleotidic impurities

HPD and crude heparin samples were evaluated by using High Performance Liquid

Chromatography (HPLC), applying the method “Nucleotidic impurities” described in the monograph

section of heparin sodium from United States Pharmacopoeia (USP), by using USP Adenosine

reference standard (U.S. Pharmaceutical Convention, Rockville, USA, Cat. No. 1012123) [3].

Before running the HPLC analysis, we treated samples by using 10 µL benzonase (Sigma-Aldrich,

Saint Louis, USA, Cat. No. E1014), 222 U alkaline phosphatase (Sigma-Aldrich, Cat. No P4978)

and 125 µL phosphodiesterase I solution (0.1U/µL, Worthington, Lakewood, USA, Cat. No. VPH),

as clearly described in [3]. We performed the HPLC analysis by using the Agilent Infinity 1260

(Agilent Technologies, Santa Clara, USA), with LC Mode, 4,6 mm x 15 cm column (4-µm packing

L1, from Sigma-Aldrich,), 260 nm UV detector and the following temperatures: 37±1°C for the

autosampler and 18°C for the column. The flow rate is fixed as 1 mL/min, with an injection volume

of 10 µL and the runtime fixed to 25 min. Adenosine USP standard was injected six times to control

reproducibility. Then, other retention times were set up by using the table of relative retention time

from USP [3].

Glycogen treatment, DNA extraction and purification

Glycogen treatment of 1 h was performed, by using glycogen from Mytilus edulis (Sigma-Aldrich),

following manufacturer’s procedure. The starting amount of HPD was 25 mg, dissolved in 100 µL

of molecular biology grade water. Finally, pellets were resuspended in molecular biology grade

water.

DNA extraction was performed by using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany),

according to manufacturer’s protocols. 4 µL internal control DNA (Primer DesignTM Ltd.,

Southampton, UK) were added to HPD and crude heparin samples before the DNA extraction,

together with the addition of proteinase K, as indicated by manufacturer.


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After extraction, DNA was purified by using DNeasy PowerClean Pro Cleanup kit (Qiagen),

according to manufacturer’s instructions, with a 50 µL final elution with molecular biology grade

water.

DNA amplification

Multiplex Real-Time PCR was performed by using the Light Cycler 480 thermal cycler (Roche

Diagnostics, Basel, Switzerland), LightCycler 480 Probe Master (Roche Diagnostics) and

Speciation standard kit of Bos taurus, Capra hircus, Ovis aries and Sus scrofa (Primer DesignTM

Ltd.), according to manufacturer’s instructions. The Real-Time PCR mixture of 20 µL included: 10

µL Probe Master, 1 µL species-specific primer and probe (FAM, 465-510 nm) mix, 1 µL internal

control DNA primer and probe (VIC, 533-580 nm) mix, 3 µL molecular biology grade water and,

finally, 5 µL DNA from treated samples, positive control or negative control (molecular biology

grade water). Amplified sequences are patented and minimum information for publication from the

supplier is summarized in Table S1 (see Electronic Supplementary Material, ESM). The thermal

protocol was set up as follows: 10 min at 95°C for initial denaturation, 50 cycles of denaturation (10

s at 95°C) and annealing/extension (1 min at 60°C, with fluorescence detection) and, extension (15

s at 78°C), and, finally, 30 s at 40°C to cool the plate.

Positive control DNA is different for each tested species and it is constituted by known copy

numbers of the amplified sequence for each species. Reaction conditions worked correctly only if

species-specific positive control DNA crossing point (Cp) is included in the range 16-23

amplification cycles (cs) at the end of the run, as mentioned in the manufacturer’s protocol. Internal

control DNA works as a positive control for the extraction and purification processes, to assess

whether DNA polymerase inhibitors have been correctly removed. Kits from Primer Design

contains a separate mix with specific primers and probe for the detection of such exogenous DNA,

which is not species-specific. The different emission ranges of the two probes, FAM and VIC, allow

performing a multiplex reaction. As already mentioned, such internal control DNA was spiked into

heparinoid sample before the DNA extraction process. Extraction protocol works correctly and

inhibitors are removed when internal control DNA has a Cp within the range 25-31 cs. The

Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler

480 II software database was applied during analysis.

Statistical analysis

Quantitative variables between groups were analyzed by different statistical analyses, depending

on the sample types (performed statistical test is indicated in the caption of each figure). P values

<0.05 were considered statistically significant. Data shown in the graphs are represented as the

mean±SEM (Standard Error of the Mean). Statistical analysis were performed using Prism 8.1


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(Graphpad Software Inc., La Jolla, USA). Normality analysis was performed before statistical

comparisons.

RESULTS

Crude heparin displays higher DNA amount than HPD

Samples of crude heparin and HPD were analyzed by optical density at 260 nm. Results are

shown in table of Fig. 1, reported as absorbance values (ABS, expressed as AU). Sample analysis

to estimate the DNA amount reported high quantity of nucleotides in crude heparins (mean ABS =

1.19±0.07 AU), and, on the other side, lower amounts in HPDs (mean ABS = 0.33±0.01 AU), with

a statistically significant variation (P < 0.0001, Mann-Whitney test), as reported in Fig. 1.

Absorbance values of HPD samples from different suppliers (HPD-X and HPD-Y) are reported in

the table of Fig. 1.

Moreover, different DNA amounts in one sample of crude heparin and one HPD (from Y supplier)

sample were evaluated with a chromatographic analysis of nucleotidic impurities. We observed

several peaks, identified as specific nucleotides, in the crude heparin sample (Fig. 2, orange line),

while in the HPD (Y) sample (Fig. 2, black line) observed peaks presented lower intensity.

Retention times of nucleotidic impurities are reported in table of Fig. 2. Other peaks were not

considered since they were also observed in blank samples. Overall, nucleotidic impurity

percentage in crude heparin was equal to 2.15 % m/m, while HPD (Y) sample presented 0.14 %

m/m.

As displayed in Fig. 3, crude heparin (B) showed higher DNA amount than HPD samples, also

after Real-Time PCR, since the mean Cp for Sus scrofa was 18.81±0.1 cs, similar to the porcine

positive control (A, mean Cp = 19.83±0.04 cs). On the other side, three HPD samples (C) reported

mean Cp = 32.84±0.30 cs (33.44, 33.19 and 31.90 cs, respectively) after DNA extraction and

purification. Negative control (D) was compliant.

DNA purification is crucial to remove inhibitory effect of the heparin on DNA polymerase

Fig. S1 (see ESM) reports the amplification curves of the HPD-Y sample that underwent DNA

extraction and purification (B), and DNA extraction only (C), besides positive control (A, mean Cp =

17.94±0.13 cs). While purified DNA reported positive mean Cp = 29.72±0.01 cs, the extracted-only

DNA showed no Cp, as the negative control (D).

The analysis of internal control DNA, reported in Fig. S2 (see ESM), was performed on the same

samples displayed in Fig. S1 (see ESM). Here, we show that DNA amplification occurs only in

extracted-and-purified samples (A, mean Cp = 29.63±0.03 cs), while extracted-only DNA (B)

presents an amplification curve similar to the negative control (C).


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DNA detection increases after glycogen treatment

Fig. 4 shows Mean Cp values of 1-h glycogen-treated (+) or untreated (-) HPD samples after DNA

amplification with Sus scrofa specific primers on 11 HPD samples, 4 HPD-X and 7 HPD-Y. The

glycogen treatment allows gaining 1 cycle when we performed Real-Time PCR (P= 0.0066, paired

t-test): mean Cpuntreated was 32.09±0.60 cs, while mean Cptreated was 31.04±0.55 cs. Similarly, table

in this figure reports the mean Cp values of amplified swine DNA from 11 HPD samples, with or

without 1-h glycogen treatment, calculated from two replicates for each sample.

Amplification curves (reported in ESM Fig. S3) show an increase in detected DNA amounts,

comparing samples treated with glycogen for 1 hour, before extracting and purifying DNA (C, mean

Cp = 29.72±0.01 cs), and untreated ones (D, extraction and purification only), with mean Cp =

32.15±0.31 cs. Extending the glycogen treatment to 16 hours (B, overnight), we reported mean Cp

= 28.69±0.03 cs, confirming the increase of DNA amounts. Positive control (A, Cp = 17.94±0.13

cs) and negative control (E) complied.

Real-Time PCR validation method

According to European Pharmacopoeia (EP) [16], we validated our Real-Time PCR-based

qualitative method for the specificity, robustness and detection limit. Method validation was

performed by using positive control DNA supplied from Primer Design, as template for specificity,

robustness and detection limit. After, ten batches of HPDs from the two suppliers were analyzed to

validate this method on HPDs.

Specificity

We tested the specificity of our method, by evaluating whether primers and probes can only detect

the species expected to be present. For each primer kit of the analyzed species, three replicates

were loaded with positive control DNA from four species in the same well. Swine-specific primer kit

amplified Sus scrofa positive control (Cp =18.39±0.05 cs), while other species-specific positive

controls were not amplified (Fig. 5A). Bos taurus positive control presented Cp = 17.95±0.04 cs,

after amplification with bovine-specific primers. They also amplified other species-specific positive

control: Cp Sus scrofa = 42.14 cs; Cp Ovis aries = 40.56±1.29 cs; Cp Capra hircus = 39.42 cs. For

Sus scrofa and Capra hircus, only one replicate out of three was detectable, with a positive

crossing point, while Ovis aries DNA amplification occurred in two samples out of three, as

displayed in Fig. 5B. Amplification of Ovis aries positive control by using ovine-specific primers

reported Cp = 16.11±0.03 cs, while positive control from other species did not display any

amplification (Fig. 5C). Similarly, Capra hircus positive control was amplified by using caprine-

specific primer kit, with Cp = 17.18±0.05 cs (Fig. 5D). Amplification of such positive controls from


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other species with these primers did not show significant amplification. Specificity test results are

summarized in Table S2 (see ESM).

Robustness

EP defined the robustness as “the capacity of an analytical procedure not to be affected by

deliberate, although small, variations in parameters” [16]. This indicates the reliability of such

analytical procedure during normal usage. Our test was set as follows: Probe Master volume was

changed by 5% more and less than the correct volume (5.25 and 4.75 µL, respectively), to verify

whether such changes affect the reliability of the analysis. We chose to change Probe Master

volume, since this is the only reagent outside the standardized primer kits from Primer Design, as

shown in Fig. S4 (see ESM), after the amplification of sample DNA, we reported 32.68±0.02 cs as

mean Cp with 5 µL volume of Probe Master (B), 32.92±0.06 cs when volume was reduced to 4.75

µL (C), and, finally, 32.55±0.07 cs with 5.25 µL volume of Probe Master (D). Analysis of positive

control DNA complies (A). The percentage variation of mean Cp was 0.73% and 0.40% with 5%

volume decrease and 5% volume increase, respectively.

Moreover, cross-contamination must be evaluated, to demonstrate robustness: it is defined as a

test in which 20 alternate samples of negative samples and positive samples were loaded side-by-

side (as reported in the table in ESM Fig. S5). All negative samples revealed as negative, while all

positive controls displayed a Cp included in the range 16-23 cs, as reported in ESM Fig. S5.

Detection Limit

Detection Limit is defined as the lowest detectable amount of nucleic acid in an analyzed sample.

Such amount could be not necessarily accurately quantitated. Although the manufacturer of the

primer kits provided a fixed Detection Limit of 35 cs, we set up our Detection Limit, as the DNA

concentration that reported significant amplification in the 95% of analyzed samples with Real-

Time PCR, as requested by pharmacopeia. After the amplification of species-specific positive

controls, Sus scrofa, Bos taurus and Ovis aries primer kits reported the Detection Limit of 1.2 DNA

copies/µL (respectively, mean Cp was 38.55±0.36, 35.46±0.21 and 36.55±0.16 cs), while the

Detection Limit of Capra hircus primers was 0.6 DNA copies/µL, with 37.21±0.25 as mean Cp.

Data reported in Table 1 refer to the fixed detection limits for each species-specific kit. Other

concentrations, higher or lower, do not satisfy the aforementioned criteria.

The bovine specificity test results reported a critical issue, concerning the amplification of DNA

from other species by using Bos taurus primers, as reported in Fig. 5. Fixed detection limits for Sus

scrofa, Ovis aries and Capra hircus DNA were lower than the observed values (Cp = 42.14 cs, Cp

= 40.56±1.29 cs and Cp = 39.42 cs, respectively). Therefore, these values were considered as

nonspecific amplifications.


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HPD samples from different suppliers display different DNA amounts, after Real-Time PCR

We analyzed HPD samples from two different suppliers, by using UV spectrometry and Real-Time

PCR. On one hand, as displayed in Fig. 6, ten HPDs from the first supplier (arbitrarily named HPD-

X) show an increasing trend in absorbance values (mean absorbance = 0.35±0.01) compared to

ten samples from the second supplier (arbitrarily named HPD-Y, mean absorbance = 0.31±0.02),

without statistical significance. On the other hand, Real-Time PCR analysis reported a statistically

significant variation of crossing point, between the two suppliers: mean Cp of HPD-X samples was

28.30±1.16 cs, while HPD-Y samples reported 31.14±0.61 cs as mean Cp (P = 0.0475, Welch’s t-

test). Notably, for routine PCR analysis positive, negative and internal control were loaded on each

plate and they always complied.

DISCUSSION

Heparin and sulodexide (heparin-like product largely used as anti-coagulant drug) are

heterogeneous mixtures that cannot be entirely characterized [1, 6, 11]. Therefore, great attention

must be paid to their traceability and quality [1]. Besides BSE sanitary risk, bovine heparin has also

been associated with a higher risk of heparin-induced thrombocytopenia than porcine heparin [1,

17]. Differences in chemical structures occur mostly among porcine and bovine heparins, by using

NMR technique, since they slightly differ in the chemical structure. From a therapeutic point of

view, these slight differences result in clearly distinct drug effects [18].

Crude heparin and HPD respectively represent the raw material for heparin and sulodexide

manufacturing processes. In pharmacopeia, a monograph is dedicated only to heparin, but not for

sulodexide nor HPDs [2-4]. Monographs indicate the PCR as the elective technique to determine

origin species. However, Hydrogen Nuclear Magnetic Resonance (H-NMR) is the widely used

method to discriminate the porcine or the bovine origin of heparin, HPDs and sulodexide. However,

by using this technique, a part of the possible contaminants could be hidden by the heparin spectra

[11]. Moreover, standards for the origin determination of other ruminant species, e.g. ovine and

caprine, are not commercially available so far. Finally, such method does not ensure high

sensitivity, since declared purity of swine origin for H-NMR method for origin determination is 94%

in our experience.

In this study, we showed a qualitative method for the detection of species-specific DNA, by using

Real-Time PCR, to evaluate possible contamination from three of the most common ruminant

species in heparinoids. Notably, through this technique, which detects the presence of DNA rather

than the differences in chemical structures, we overcome the critical issue encountered with H-

NMR analysis, as previously mentioned.


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First, we performed UV spectrometry analysis to evaluate differences in DNA amounts in crude

heparin and HPD with low grade of sensitivity: crude heparin displays higher nucleic acid levels

compared to HPD (P < 0.0001). Indeed, HPDs manufacturing process requires more steps than

crude heparin one: this determines, in the end, a substantial reduction of DNA amounts, as

confirmed by our results. After, we performed HPLC analysis to better investigate the relative

abundance of DNA amounts, detected as nucleotidic impurities, in crude heparin compared to

HPDs, with higher levels of sensitivity and reliability. By using HPLC, we confirmed the data

observed with UV spectrometry: crude heparin contains higher DNA amounts respect to HPD.

Finally, also Real-Time PCR analysis revealed very higher DNA amounts in crude heparin if

compared to heparinoid samples, with a difference of 14 amplification cycles among those two

sample types.

DNA detection in HPD samples was possible only after the introduction of different steps, before

and after the DNA extraction, because it alone does not remove all the heparin residues and does

not allow detecting significant DNA amounts by using Real-Time PCR and, therefore, we inserted

DNA purification after its extraction. Notably, the presence of heparin residues heavily interferes

with DNA polymerase [15, 19]. Although we performed DNA purification, we inserted internal

control DNA in the PCR plate, to ascertain possible heparin interference. Moreover, DNA

extraction method is optimized by using proteinase K, which reduces the viscosity of the dissolved

HPD in water, because of its action on the proteins from the intestinal mucosa. As previously

demonstrated, our HPD samples characterize for low DNA amounts. Therefore, we evaluated the

positive impact of glycogen treatment before the DNA extraction on HPDs. Notably, glycogen is an

inert carrier that traps nucleic acids and, by using it, it is possible to recover oligonucleotides as

short as eight base pairs and low amounts of DNA/RNA. Glycogen forms a clearly visible pellet

during ethanol precipitation, since it is insoluble in this solvent. Compared to other nucleic acid

carriers, such as tRNA, linear polyacrylamide, or other methods, e.g. DNA sonication, glycogen

allows better efficiency, it is easy-to-use and it does not require particular safety precautions [20-

22]. Comparing samples treated with or without glycogen, we reported a significant difference in

mean Cp (P = 0.0066), confirming that glycogen treatment allows better detection of low DNA

amounts. Notably, enhancing the gap between crossing point of HPD samples and the threshold of

35 cycles by using glycogen treatment (as specified by kit’s manufacturer), allows a better

detection and analysis, since such threshold represents a first detection limit, as reported in the

manufacturer’s protocol of the species-specific primers. Additionally, manufacturer’s protocol

suggests incubating samples with glycogen for 1 hour or overnight. In both cases, we reported

greater detection in glycogen-treated samples, compared to untreated ones. Although overnight

treatment allows us to gain 1 cycle respect to 1-hour treatment, we chose the latter, since it is

timesaving. Thus, final sample preparation protocol, before the DNA amplification was set up as

follows: 1-h glycogen treatment, DNA extraction, DNA purification. Glycogen treatment


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concentrates nucleic acids and this reveals crucial for the correct method execution in highly

processed HPD samples.

As requested by pharmacopeia, the selected method needs to be validated. To validate qualitative

Real-Time PCR methods, EP and the US Food and Drug Administration guidance for industry

strongly recommends the evaluation of the specificity, detection limit and robustness [16, 23]. Our

method meets all these requirements, since it reveals specific and robust and with low detection

limit. Primer kits are specific, since only species-specific DNA was amplified by these primers.

Even if Bos taurus primers amplified DNA from other three species, registered crossing points

were over the detection limit and, therefore, considered as negative. Robustness is guaranteed

since no significant variations in Cp were reported, although the variation of ± 5% volume of Probe

Master. Moreover, no cross-contamination must be reported in all samples. Detection limit for all

species-specific primer kits was fixed as 1.2 DNA copies/µL, except for Capra hircus one, whose

detection limit was set up to 0.6 DNA copies/µL. For both cases, this allows highly sensitive and

efficient DNA detection. Since our Detection Limits for each species are lower than the one

declared from Primer Design, with higher grade of sensitivity and specificity for HPD.

Lastly, we aimed to characterize different suppliers of HPD, by using UV spectrometry, as well as

Real-Time PCR. Non-statistically significant differences in absorbance values among the two

suppliers, 0.35 rather than 0.31, transformed in significant variations when we performed Real-

Time PCR analysis (higher crossing point means lower DNA starting quantity). Both suppliers were

validated since the quality and the safety of their samples met the criteria requested by

pharmacopeia. We hypothesize that greater DNA fragmentation could occur in samples from the

second supplier (HPD-Y), due to possible differences in manufacturing process determining a

decrease in mean Cp, while absorbance values do not significantly change. This will be

investigated during the setup of the quantitative method.

In this study, we finally reached our proposed aim to set up a qualitative Real-Time PCR method to

certify the origin determination of HPD samples. We combined three different sequential steps:

glycogen treatment, DNA extraction and, finally, DNA purification, to ensure the greatest recovery

of DNA amount, on one hand, and the elimination of heparin from the final eluted sample, on the

other hand, in highly processed HPD samples.

In conclusion, our Real-Time PCR-based method revealed efficient and highly sensitive to assess

species origin in HPDs, which undergo long and complex manufacturing process and constitute the

starting materials of the sulodexide manufacturing process. The introduction of glycogen treatment

together with the DNA purification step after the DNA extraction, ensure DNA detection grade and

the elimination of heparin residues. Therefore, this innovative method contributes to improve

quality, safety and traceability of highly processed raw materials, as heparinoid, allowing us to add


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a checkpoint for the control of origin species during the manufacturing process of sulodexide. In

the end, this method is also useful to characterize HPDs from different suppliers.

ACKNOWLEDGEMENTS

The research was performed in the Quality Control and R&D laboratories of Biofer S.p.A., Medolla,

Italy. The authors would like to thank Senior Management of the company, and, particularly, Dr.

Alessandro Lapini Sacchetti and Dr. Gianmaria Ristori for their support, endorsement and

extensive knowledge in the field of this class of active substances.

CONFLICT OF INTEREST

The authors declare and disclose that no direct relationships or interests can potentially influence

or impart bias on the work.

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4. European Directorate for the Quality of Medicine & HealthCare. Heparin sodium. In: European Pharmacopoeia 9.0. Strasbourg; 2017. pp: 2644-2646; Volume II.

5. Bouchard O, Hoppensteadt D, Maia P, de Castro AS, Kumar E, Guler N, Jeske W, Kahn D, Walenga JM, Coyne E, Yao Y, Fareed J. Porcine and ovine mucosal heparins and their depolymerized derivatives are comparable in contrast to their bovine equivalents. Blood. 2016. 2016;128(22):5027.

6. Carroll BJ, Piazza G, Goldhaber SZ. Sulodexide in venous disease. J Thromb Haemost. 2019;17(1):31-8. doi:10.1111/jth.14324.

7. Veraldi N, Guerrini M, Urso E, Risi G, Bertini S, Bensi D et al. Fine structural characterization of sulodexide. J Pharm Biomed Anal. 2018;156:67-79. doi:10.1016/j.jpba.2018.04.012.

8. Coccheri S, Mannello F. Development and use of sulodexide in vascular diseases: implications for treatment. Drug Des Devel Ther. 2013;8:49-65. doi:10.2147/DDDT.S6762.

9. Concannon SP, Wimberley PB, Workman WE. A quantitative PCR method to quantify ruminant DNA in porcine crude heparin. Anal Bioanal Chem. 2011;399(2):757-62. doi:10.1007/s00216-010-4362-8.

10. Tanabe S, Hase M, Yano T, Sato M, Fujimura T, Akiyama H. A real-time quantitative PCR detection method for pork, chicken, beef, mutton, and horseflesh in foods. Biosci Biotechnol Biochem. 2007;71(12):3131-5. doi:10.1271/bbb.70683.


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11. Auguste C, Dereux S, Martinez C, Anger P. New developments in quantitative polymerase chain reaction applied to control the quality of heparins. Anal Bioanal Chem. 2011;399(2):747-55. doi:10.1007/s00216-010-4232-4.

12. Sakai Y, Kotoura S, Yano T, Kurihara T, Uchida K, Miake K et al. Quantification of pork, chicken and beef by using a novel reference molecule. Biosci Biotechnol Biochem. 2011;75(9):1639-43. doi:10.1271/bbb.110024.

13. Ding M, Bullotta A, Caruso L, Gupta P, Rinaldo CR, Chen Y. An optimized sensitive method for quantitation of DNA/RNA viruses in heparinized and cryopreserved plasma. J Virol Methods. 2011;176(1-2):1-8. doi:10.1016/j.jviromet.2011.05.012.

14. Huang Q, Yao CY, Chen B, Wang F, Huang JF, Zhang X et al. Species-specific identification by inhibitor-controlled PCR of ruminant components contaminating industrial crude porcine heparin. Mol Cell Probes. 2006;20(3-4):250-8. doi:10.1016/j.mcp.2006.01.005.

15. Yokota M, Tatsumi N, Nathalang O, Yamada T, Tsuda I. Effects of heparin on polymerase chain reaction for blood white cells. J Clin Lab Anal. 1999;13(3):133-40.

16. European Directorate for the Quality of Medicine & HealthCare. Nucleic acid amplification techniques. In: European Pharmacopoeia 9.0. Strasbourg; 2017. pp: 214-219; Volume I.

17. Houiste C, Auguste C, Macrez C, Dereux S, Derouet A, Anger P. Quantitative PCR and disaccharide profiling to characterize the animal origin of low-molecular-weight heparins. Clin Appl Thromb Hemost. 2009;15(1):50-8. doi:10.1177/1076029608320831.

18. Tovar AM, Santos GR, Capille NV, Piquet AA, Glauser BF, Pereira MS et al. Structural and haemostatic features of pharmaceutical heparins from different animal sources: challenges to define thresholds separating distinct drugs. Sci Rep. 2016;6:35619. doi:10.1038/srep35619.

19. Beutler E, Gelbart T, Kuhl W. Interference of heparin with the polymerase chain reaction. Biotechniques. 1990;9(2):166.

20. Helms C, Graham MY, Dutchik JE, Olson MV. A new method for purifying lambda DNA from phage lysates. DNA. 1985;4(1):39-49. doi:10.1089/dna.1985.4.39.

21. Hengen PN. Carriers for precipitating nucleic acids. Trends Biochem Sci. 1996;21(6):224-5.

22. Tracy S. Improved rapid methodology for the isolation of nucleic acids from agarose gels. Prep Biochem. 1981;11(3):251-68. doi:10.1080/00327488108061767.

23. Ekins J, Peters SM, Jones YL, Swaim H, Ha T, La Neve F et al. Development of a multiplex real-time PCR assay for the detection of ruminant DNA. J Food Prot. 2012;75(6):1107-12. doi:10.4315/0362-028X.JFP-11-415.


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190x275mm (96 x 96 DPI)


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FIGURE CAPTIONS

Figure 1 Histograms report the mean absorbance at UV wavelength of 260 nm, expressed as Absorbance Units (AU), of 10 samples of crude heparin (CH, 1.19±0.07 AU) and 20 samples of heparinoids (HPDs, 0.33±0.01 AU). A significant variation in absorbance was observed comparing crude heparin with HPDs (****: P < 0.0001, Mann-Whitney test). Table reports the absorbance values for each sample and mean Absorbance values.

Figure 2 Overlay of chromatograms of crude heparin (orange line) and heparinoid (HPD-Y, black line), after analysis of nucleotide impurities by using HPLC [3]. Table reports the retention times of nucleotide impurities in crude heparin and heparinoid, identified from the relative retention times supplied by United States Pharmacopoeia. We observed several peaks, identified as specific nucleotides, in the crude heparin sample (nucleotide impurities: 2.15% m/m), while in the HPD sample detected peaks presented lower intensity (nucleotide impurities: 0.14% m/m).

Figure 3 Typical Real-Time PCR profile of the Sus scrofa DNA from positive control (A, red lines, Cp: 19.83±0.04 cs), crude heparin (B, blue lines, Cp: 18.81±0.1 cs), three different heparinoid samples from one supplier (HPD-Y, C, green, yellow and brown lines, Cp: 32.84±0.30 cs; 33.44, 33.19 and 31.90 cs, each) and negative control (D, black lines). A 14-fold increase in mean Cp was observed comparing crude heparin and HPD-Y samples. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles.

Figure 4 The graph reports the crossing points of amplified swine DNA from 11 heparinoid samples with (+) or without (-) glycogen treatment of 1 hour, 4 from the first supplier (HPD-X) and 7 from the other supplier (HPD-Y). Mean Cp values, expressed as amplification cycles, of Sus scrofa DNA, with or without 1-h glycogen treatment, were calculated from two replicates for each sample. Green lines report a decrease in sample Cp, while red lines show an increase in Cp after glycogen treatment. At the bottom of the table, we report mean Cp±SEM values from all the HPD samples, with or without 1-h glycogen treatment. We reported a significant decrease of Cp in samples treated with glycogen for 1 h, compared to untreated ones (**: P < 0.01, paired t-test ), regardless of the supplier. Data are expressed as mean Cp (cs). DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: crossing point; cs: cycles; SEM: Standard Error of the Mean.

Figure 5 Specificity test results of each species-specific primer kit: Sus scrofa (A, grey lines, Cp: 18.39±0.05 cs), Bos taurus (B, blue lines, Cp: 17.95±0.04 cs), Ovis aries (C, red lines, Cp: 16.11±0.03 cs) and Capra hircus (D, green lines, Cp: 17.18±0.05 cs). For each test, DNA positive control of each species and negative control were loaded. While porcine, ovine and caprine-specific primers only amplified species-specific DNA positive control, bovine-specific primers (B) reported amplification of porcine (Cp: 42.14 cs, grey line), ovine (Cp: 40.56±1.29 cs, red lines) and caprine (Cp: 39.42 cs, green line) DNA, with Cp>35 cs. DNA amplification was performed by using the four species-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles.

Figure 6 Absorbance at UV wavelength of 260 nm, expressed as AU, and Cp values, expressed as cs, for 20 heparinoid samples, 10 from one supplier (HPD-X) and 10 from the other (HPD-Y). Table in the figure reports the mean crossing point value of each sample. Histograms report the mean absorbance (A) and mean Cp (B) of HPDs from the two suppliers. While we reported no significant variation in absorbance, mean Cp from HPD-Y was significantly higher than the one from HPD-X


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(*: P< 0.05, Welch’s t-test). DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. AU: Absorbance Units; Cp: Crossing point; cs: cycles.

TABLE CAPTIONS

Table 1 Fixed detection limit for all the tested species-specific primer kits, expressed as copies of DNA positive control in 1 µL. Reported data show the Cp, expressed as cs, of three samples treated for 1 h with glycogen, extracted and purified, after three independent dilution series. Mean Cp and SEM are reported on the right of the table. DNA amplification was performed by using the four species-specific primer kits. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: amplification cycles; SEM: Standard Error of the Mean.


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Fig. 1

Absorbance at 260 nm (AU)Crude Heparin (CH)

CH-01 CH-02 CH-03 CH-04 CH-05 CH-06 CH-07 CH-08 CH-09 CH-10 Mean1.2 1.2 1.3 1.2 1.2 1.2 1.3 1.1 1.1 1.1 1.19±0.07

Heparinoid (HPD)

HPD-X01 HPD-X02 HPD-X03 HPD-X04 HPD-X05 HPD-X06 HPD-X07 HPD-X08 HPD-X09 HPD-X10 Mean0.34 0.40 0.41 0.41 0.36 0.36 0.30 0.32 0.28 0.31

0.33±0.01HPD-Y01 HPD-Y02 HPD-Y03 HPD-Y04 HPD-Y05 HPD-Y06 HPD-Y07 HPD-Y08 HPD-Y09 HPD-Y100.33 0.35 0.34 0.25 0.24 0.21 0.31 0.35 0.34 0.40


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Fig. 2

Res

pons

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Time (min)

CRUDE HEPARIN (Nucleotide impurities: 2.15% m/m)

HEPARINOID (Nucleotide impurities: 0.14% m/m)

C dC

U

mdC

G

dGT

A

dA

Retention time (min)Nucleotide USP relative retention time Crude Heparin Heparinoid

Cytidine (C) 0.28 3.490 3.476Deoxycytidine (dC) 0.38 4.597 4.574

Uridine (U) 0.40 4.818 4.8005-metyl-2-deoxycytidine (mdC) 0.66 9.044 8.977

Guanosine (G) 0.81 10.116 10.0602-deoxyguanosine (dG) 0.89 11.462 11.446

Thymidine (T) 0.92 12.309 12.326Adenosine (A) 1.00 13.161 13.167

2-deoxyadenosine (dA) 1.04 13.473 13.500


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Fig. 3Fl

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Fig. 4

1-h glycogen treatment

- + Mean (cs) Mean (cs)

HPD-X07 33.60 32.37HPD-X08 32.91 32.56HPD-X09 32.57 33.14HPD-X10 32.66 31.58HPD-Y05 29.51 29.13HPD-Y06 30.05 29.28HPD-Y07 30.68 28.56HPD-Y08 29.31 29.59HPD-Y09 35.63 33.64HPD-Y10 33.96 31.88HPD-Y11 32.15 29.72

Mean±SEM 32.09±0.60 31.04±0.55


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Fig. 5

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A

B

Fig. 6

UV spectroscopy Real-Time PCRSample ABS 260 nm (AU) Mean ABS SEM Cp (cs) Mean Cp SEM

HPD-X01 0.34

0.35 0.01

27.46 27.28

28.30 1.16

HPD-X02 0.40 25.13 25.06HPD-X03 0.41 24.89 24.84HPD-X04 0.41 24.26 24.26HPD-X05 0.36 25.98 25.94HPD-X06 0.36 25.78 25.73HPD-X07 0.30 32.48 32.26HPD-X08 0.32 32.57 32.55HPD-X09 0.28 33.41 32.87HPD-X10 0.31 31.55 31.61HPD-Y01 0.33

0.31 0.02

33.46 33.41

31.14 0.61

HPD-Y02 0.35 33.27 33.10HPD-Y03 0.34 32.01 31.79HPD-Y04 0.25 30.75 30.84HPD-Y05 0.24 29.11 29.14HPD-Y06 0.21 29.26 29.29HPD-Y07 0.31 28.59 28.52HPD-Y08 0.35 29.56 29.61HPD-Y09 0.34 33.61 33.67HPD-Y10 0.40 31.94 31.81


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Table 1

SpeciesDetection Limit

(DNA copies/µL)

Crossing point, glycogen-treated DNA positive control (cycles) Mean Cp SEM

Sus scrofa 1.2 38.01 38.93 38.91 40.01 37.95 37.42 37.77 40.44 37.55 38.55 0.36Bos taurus 1.2 35.65 35.31 34.70 35.40 34.95 35.94 35.09 36.81 35.29 35.46 0.21Ovis aries 1.2 36.72 36.12 35.79 36.07 36.98 36.81 36.92 37.22 36.28 36.55 0.16

Capra hircus 0.6 38.01 35.91 37.79 36.78 38.43 36.58 36.85 37.60 36.91 37.21 0.25


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Analytical and Bioanalytical Chemistry

Electronic Supplementary Material

Highly sensitive real-time PCR method to identify species origin

in heparinoids

Simone Pecorini, Giulio Camurri, Lucia Torrini, Roberta Ferraresi


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Fig. S1 Typical Real-Time PCR profile of the Sus scrofa DNA from positive control (A, red lines, Cp: 17.94±0.13 cs), heparinoid (HPD-Y, second supplier) after 1-h glycogen treatment, extraction and purification (B, green lines, 29.72±0.01 cs), the same heparinoid after 1-h glycogen treatment and extraction without purification (C, blue lines) and negative control (D, black lines). DNA amplification occurred only in extracted-and-purified samples, while extracted-only DNA presented an amplification curve similar to the negative control. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles


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Fig. S2 Typical Real-Time PCR profile of the internal control DNA after extraction and purification of the same HPD-Y sample analyzed in Figure 3 and 4 (A, brown lines, Cp: 29.63±0.03 cs), after extraction only (B, green lines) and negative control (C, grey lines). While glycogen-treated, extracted and purified DNA reported positive amplification, the glycogen-treated and extracted DNA showed no Cp. DNA amplification was performed by using the internal control-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles


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Fig. S3 Typical Real-Time PCR profile of the Sus scrofa DNA from positive control (A, red lines, Cp: 17.94±0.13 cs), heparinoids after overnight glycogen treatment, extraction and purification (B, green lines, Cp: 28.69±0.03 cs), heparinoids after 1-h glycogen treatment, extraction and purification (C, blue lines, Cp: 29.72±0.01 cs), heparinoids without glycogen treatment (D, yellow lines, Cp: 32.15±0.31 cs) and negative control (E, black lines). Although after the overnight glycogen treatment Cp was lower than the one after 1-h treatment, the latter determined a decrease in Cp, if compared to the extracted-and-purified sample. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles


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Probe Master mix volume (µL)4.75 (C) 5.00 (B) 5.25 (D)

Cp (cs) 32.98 32.69 32.6232.86 32.66 32.48

Mean Cp (cs) 32.92 32.68 32.55SEM (cs) 0.06 0.02 0.07

% variation 0.73% 0.40%

Fig. S4 Results of robustness test performed with Sus scrofa primer kit. We performed robustness test by modifying the probe master mix volume: amplification curves of DNA positive control (A, blue lines), samples by using standard volume (B, grey lines, Cp: 32.68±0.02), by using 5%-reduced-volume (C, red lines, Cp: 32.92±0.06 cs) and 5%-increased volume (D, green lines, Cp: 32.05±0.07 cs) of probe master mix. 5%-variation of probe master mix volume reflected in slight variations in mean Cp (0.73% and 0.40%), confirming the robustness of the method. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles


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Sus scrofaBos taurusOvis ariesCapra hircusNegative control

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1 2 3 4 5 6 7 8 9 10 11 12

ABlank Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus Pos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra Pos Ctr Capra Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus

- - 16.88 - 18.30 - 18.63 - 16.62 - 18.16 -

BPos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra Pos Ctr Capra Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus Pos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra

19.26 - 18.99 - 17.44 - 18.50 - 19.51 - 19.43 -

CPos Ctr Capra Neg Ctr Capra

17.90 -

Fig. S5 Results of cross-contamination test, as Cp. We loaded more than 20 samples, alternating negative and DNA positive control samples in one Real-Time PCR microplate. Reaction conditions worked correctly only if the amplification of species-specific positive control DNA crossing point occurred in the range 16-23 cs, as mentioned in the manufacturer’s protocol. All negative samples revealed as negative, while all positive samples presented Cp included in the correct range. Therefore, cross-contamination was successfully tested. DNA amplification was performed by using the four species-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: crossing point; cs: amplification cycles


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Table S1 Accession numbers, context sequence lengths and anchor nucleotides for each species-specific primer used in the DNA amplification (Primer DesignTM Ltd.). bp: base pairs

Accession Number Context Sequence Anchor NucleotideSus scrofa AF034253.1 195 bp 14549

Bos taurus NC_006853 208 bp 15931

Ovis aries HM236178.1 106 bp 14872

Capra hircus KM998968.1 123 bp 8037

Table S2 Results of the specificity test for each species-specific primer kit. Data report the mean Cp of DNA positive control from all species, after amplification with each species-specific primer kit, expressed as cs. By using bovine species-specific primers, positive amplification occurred only in one sample of Sus scrofa (Cp: 42.14 cs) and Capra hircus (Cp: 39.42 cs) DNA positive control, instead of two (as displayed for Ovis aries, Cp: 40.56±1.29 cs). This explains why single values were reported for these two species. DNA amplification was performed by using the four species-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: amplification cycles

Mean crossing point, by using species-specific primer kits (±SEM, cycles)

Sus scrofa Bos taurus Ovis aries Capra hircus

Sus scrofa 18.39±0.05 42.14 - -

Bos taurus - 17.95±0.04 - -

Ovis aries - 40.56±1.29 16.11±0.03 -

DNA

posit

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cont

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Capra hircus - 39.42 - 17.18±0.05


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Analytical and Bioanalytical Chemistry

Electronic Supplementary Material

Highly sensitive real-time PCR method to identify species origin

in heparinoids

Simone Pecorini, Giulio Camurri, Lucia Torrini, Roberta Ferraresi


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Fig. S1 Typical Real-Time PCR profile of the Sus scrofa DNA from positive control (A, red lines, Cp: 17.94±0.13 cs), heparinoid (HPD-Y, second supplier) after 1-h glycogen treatment, extraction and purification (B, green lines, 29.72±0.01 cs), the same heparinoid after 1-h glycogen treatment and extraction without purification (C, blue lines) and negative control (D, black lines). DNA amplification occurred only in extracted-and-purified samples, while extracted-only DNA presented an amplification curve similar to the negative control. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles


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Fig. S2 Typical Real-Time PCR profile of the internal control DNA after extraction and purification of the same HPD-Y sample analyzed in Figure 3 and 4 (A, brown lines, Cp: 29.63±0.03 cs), after extraction only (B, green lines) and negative control (C, grey lines). While glycogen-treated, extracted and purified DNA reported positive amplification, the glycogen-treated and extracted DNA showed no Cp. DNA amplification was performed by using the internal control-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles


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Fig. S3 Typical Real-Time PCR profile of the Sus scrofa DNA from positive control (A, red lines, Cp: 17.94±0.13 cs), heparinoids after overnight glycogen treatment, extraction and purification (B, green lines, Cp: 28.69±0.03 cs), heparinoids after 1-h glycogen treatment, extraction and purification (C, blue lines, Cp: 29.72±0.01 cs), heparinoids without glycogen treatment (D, yellow lines, Cp: 32.15±0.31 cs) and negative control (E, black lines). Although after the overnight glycogen treatment Cp was lower than the one after 1-h treatment, the latter determined a decrease in Cp, if compared to the extracted-and-purified sample. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles


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C

D

Probe Master mix volume (µL)4.75 (C) 5.00 (B) 5.25 (D)

Cp (cs) 32.98 32.69 32.6232.86 32.66 32.48

Mean Cp (cs) 32.92 32.68 32.55SEM (cs) 0.06 0.02 0.07

% variation 0.73% 0.40%

Fig. S4 Results of robustness test performed with Sus scrofa primer kit. We performed robustness test by modifying the probe master mix volume: amplification curves of DNA positive control (A, blue lines), samples by using standard volume (B, grey lines, Cp: 32.68±0.02), by using 5%-reduced-volume (C, red lines, Cp: 32.92±0.06 cs) and 5%-increased volume (D, green lines, Cp: 32.05±0.07 cs) of probe master mix. 5%-variation of probe master mix volume reflected in slight variations in mean Cp (0.73% and 0.40%), confirming the robustness of the method. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles


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For Peer Review

Fluo

resc

ence

FAM

cha

nnel

(465

-510

nm

)

Cycles

Sus scrofaBos taurusOvis ariesCapra hircusNegative control

16-23 cs

1 2 3 4 5 6 7 8 9 10 11 12

ABlank Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus Pos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra Pos Ctr Capra Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus

- - 16.88 - 18.30 - 18.63 - 16.62 - 18.16 -

BPos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra Pos Ctr Capra Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus Pos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra

19.26 - 18.99 - 17.44 - 18.50 - 19.51 - 19.43 -

CPos Ctr Capra Neg Ctr Capra

17.90 -

Fig. S5 Results of cross-contamination test, as Cp. We loaded more than 20 samples, alternating negative and DNA positive control samples in one Real-Time PCR microplate. Reaction conditions worked correctly only if the amplification of species-specific positive control DNA crossing point occurred in the range 16-23 cs, as mentioned in the manufacturer’s protocol. All negative samples revealed as negative, while all positive samples presented Cp included in the correct range. Therefore, cross-contamination was successfully tested. DNA amplification was performed by using the four species-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: crossing point; cs: amplification cycles


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For Peer Review

Table S1 Accession numbers, context sequence lengths and anchor nucleotides for each species-specific primer used in the DNA amplification (Primer DesignTM Ltd.). bp: base pairs

Accession Number Context Sequence Anchor Nucleotide Sus scrofa AF034253.1 195 bp 14549 Bos taurus NC_006853 208 bp 15931 Ovis aries HM236178.1 106 bp 14872

Capra hircus KM998968.1 123 bp 8037

Table S2 Results of the specificity test for each species-specific primer kit. Data report the mean Cp of DNA positive control from all species, after amplification with each species-specific primer kit, expressed as cs. By using bovine species-specific primers, positive amplification occurred only in one sample of Sus scrofa (Cp: 42.14 cs) and Capra hircus (Cp: 39.42 cs) DNA positive control, instead of two (as displayed for Ovis aries, Cp: 40.56±1.29 cs). This explains why single values were reported for these two species. DNA amplification was performed by using the four species-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: amplification cycles

Mean crossing point, by using species-specific primer kits (±SEM, cycles)

Sus scrofa Bos taurus Ovis aries Capra hircus

DNA

posit

ive

cont

rol Sus scrofa 18.39±0.05 42.14 - -

Bos taurus - 17.95±0.04 - -

Ovis aries - 40.56±1.29 16.11±0.03 -

Capra hircus - 39.42 - 17.18±0.05


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For Peer Review10.1007...For Peer Review 1 Highly sensitive real-time PCR method to identify species...

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