Journal of Virological Metho&, 37 (1992) 209-2 18 209 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00

VIRMET 01318

Increased sensitivity for detection of human cytomegalovirus in urine by removal of inhibitors

for the polymerase chain reaction

Yuki Yamaguchia, Takashi Hironakaa, Michiko Kajiwaraa3b, Emiko Tatenoa, Hiroshi Kitaa and Kanji Hiraia

‘Department of Cell Regulation, Division of Virology and Immunology, Medical Research Institute and bDepartment of Pediatrics,

Medical School, Tokyo Medical and Dental University. Tokyo, Japan

(Accepted 11 December 1991)

Summary

The presence of inhibitors in urine interferes with the enzymatic reaction of the polymerase chain reaction (PCR) for detection of human cytomegalovirus (HCMV). To remove inhibitors, HCMV virions in urine were precipitated with polyethylene glycol, or DNA was extracted from urine by the use of glass powder and subjected to PCR followed by Southern blot hybridization with alkaline phosphatase-linked oligonucleotide probes. These simple, rapid methods increased significantly the sensitivity of PCR for detection of HCMV in urine.

HCMV/PCR inhibitors; Urine; Southern blot hybridization; Polyethylene glycol; Glass powder

Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in fetuses and the newborn and among immuno-compromised individuals. A simple, rapid diagnosis of HCMV is required for congenital

Correspondence to: K. Hirai, Department of Cell Regulation, Division of Virology and Immunology, Medical Research Institute, Tokyo Medical and Dental University, Yushima l-S-45, Bunkyou-ku, Tokyo I 13, Japan.

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HCMV infection and various HCMV-related diseases. The standard procedure for detection of HCMV infection is isolation of HCMV from urine by a tissue culture technique. However, the disadvantage of this procedure is that it usually takes l-4 weeks. Recently, the polymerase chain reaction (PCR) technique has been used as a rapid and most sensitive method for detection of HCMV in urine and blood samples (Demmler et al., 1988; Shibata et al., 1988; Olive et al., 1989; Stanier et al., 1989; Chou et al., 1990). The significant point of PCR technique for detection of HCMV in urine is that a very small amount of urine, l-5 ~1, can be added directly to the PCR reaction mixture without purification of viral DNA. However, the presence of inhibitors in urine samples interfered with the enzyme activity of PCR (Demmler et al., 1988). The false- negative result will seriously affect the reliability of HCMV diagnosis by PCR.

This report describes the removal of PCR inhibitors from urine samples by using polyethylene glycol (PEG) and glass powder. PEG was used to precipitate HCMV in urine prior to DNA hybridization (Kimpton et al., 1990; Landini et al., 1990) while the use of glass powder for extraction of RNA or DNA was employed as a simple method to detect proviral DNA of human T- lymphotropic virus type-l (HTLV-1) and viral RNA of human immunodefi- ciency virus (HIV) in blood samples by PCR (Yamada et al., 1990). In addition, we discuss the suitability of synthetic oligonucleotides and nonradioactive oligonucleotide probes for detection of HCMV by PCR.

Materials and Methods

Urine samples

Urine samples were centrifuged at 2500 rpm for 10 min to remove the cell debris. The supernatant was directly tested for PCR amplification or pretreated with PEG and glass powder. The urine samples used here were obtained from the Department of Pediatrics at our medical school hospital and from Dr. Y. Tsutsui, Aichi Prefectural Colony, Japan. For isolation of HCMV from urine samples by tissue culture, 0.2 ml of the urine supernatant was inoculated onto human embryo lung (HEL) cells and the culture was observed for a cytopathic effect characteristic of HCMV for 4-6 weeks. Laboratory strain Towne was used as the standard HCMV strain in the experiments described here.

Precipitation of HCMV by PEG

50 ~1 of urine supernatant were mixed with 50 ~1 of 20% PEG 6000 (Wako Pure Chemical Industries, Osaka) and 25 ~1 of 2 M NaCl and incubated for 6 h on ice and centrifuged at 15 000 rpm for 30 min. The resulting precipitate was recentrifuged at 6400 rpm for 3 min to remove the supernatant as much as possible and suspended in distilled water. The suspension was subjected to PCR.

211

Extraction of HCMV DNA by glass powder

The condition for extraction of HCMV DNA from urine by glass powder was used essentially as described by Yamada et al. (1990). Briefly, 100 ~1 of urine supernatant was mixed with 100 ~1 of 6 M guanidine thiocyanic acid, 7 ~1 of 2 M NaCl and 20 ~1 of glass powder suspension (DNA PREP, Asahi Glass Co., Tokyo). After standing at room temperature for 10 min, the mixture was centrifuged at 6400 rpm for 2 min. The resulting precipitate was washed with 50% ethanol, 10 mM Tris-HCl, pH 7.4, 50 mM NaCl and centrifuged at 6400 rpm for 1 min. The washing procedure was repeated twice. Then, the DNA bound to glass powder was extracted by 50 ,ul of distilled water at 55°C for 15 min followed by centrifugation at 15 000 rpm for 2 min. A 40-~1 aliquot of the supernatant was used for PCR.

Preparation of PCR primers

Two pairs of oligonucleotide primers were synthesized in an Applied Biosystems DNA synthesizer (Foster City, CA). The first primer pair of Pl (5’- TATACCCAGACGGAAGAGAAATTCA-3’) and P2 (5’-ATAAGCCA- TAATCTCATCAGGGGAG-3’), termed MIE primers, allowed amplification of 426-bp sequences, which was chosen from exon 4 of the major immediate- early gene of HCMV (Stenberg et al., 1984). The second primer pair of P3 (5’- TGACGCGCATACATCCCGAGTACAT-3’) and P4 (5’-AT- GACGTTGCTCCGTGGAAAGAGACC-3’), termed pp71 primers, allowed amplification of the 316-bp sequence that codes for a portion of phosphory- lated protein of 71 kDa (~~71) of HCMV (Ruger et al., 1987).

PCR

Urine samples (l-30 ~1) with or without pretreatment by PEG and glass powder were boiled for 10 min, rapidly chilled in ice-water. The reaction mixture containing 1.5 mM MgC12, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1 pmol/pl of each primer, 0.001% gelatin, 200 PM each of dNTP and 0.006 units/ ~1 of Taq polymerase (AmpliTaq; Perkin-Elmer Cetus, Norwalk, CT) was added to the boiled sample to make a total volume of 20-50 ~1. Template DNA was added to the reaction mixture at a maximum concentration of 10 ng/pl. Cellular DNA used as a template was extracted as described previously (Hirai and Watanabe 1976). The amplification reaction was carried out in a DNA thermal cycler (Perkin-Elmer Cetus). Temperature cycling for PCR consisted of 94°C for 1 min, 60°C for 2 min, and 72°C for 3 min, repeated for 30 cycles, followed by 72°C for 7 min. A IO-p1 portion of the amplified product was electrophoresed in 1.8% agarose gel at 100 V using 23 mM Tris-borate buffer, pH 8.3, 0.5 mM EDTA. The bands were visualized by ethidium bromide (10 lug/ml).

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Southern blot hybridization

The conditions used for Southern blot hybridization were essentially as described previously (Hirai et al., 1984), except those for hybridization and the probe. Briefly, two oligonucleotides, probe MIE (5’-CTCCTTAATACAAGC- CATCCACATCTCCCG-3’) and probe pp7 1 (5’-GCTGTCGGTGTT- CCAAATGAACGGCCTGAT-3’) were chosen from the sequences bounded by MIE primers Pl and P2, and pp71 primers P3 and P4, respectively. These oligonucleotides were covalently cross-linked to alkaline phosphatase by Iatron Laboratories, Chiba, Japan, according to the method of Jablonski et al. (1986). A nylon filter containing immobilized DNAs was then prehybridized in 5 ml of hybridization buffer containing 0.1% sarcosine, 0.2% sodium dodecyl sulfate (SDS), 5% blocking reagent (Boeringer-Mannheim, FRG), 30% formamide and 5 x SSC (SSC = 0.15 M NaCl and 0.015 M sodium citrate) for 1 h at 37°C and then hybridized with 3 pmol of oligonucleotide-alkaline phosphatase conjugate in 5 ml of the hybridization buffer overnight at 37°C. The filter was washed 3 times with washing buffer consisting of 2 x SSC and 0.1 mM ZnClz and incubated in the washing buffer with 0.1% SDS for 45 min at 42°C. After the filter was washed 3 times with the washing buffer without SDS to remove the detergent and rinsed in 0.1 M Tris/HCl, pH 9.5, 0.1 M NaCl and 10 mM MgC12 for 1 min at room temperature, the filter was developed in 0.3 mg/ml of nitroblue tetrazolium and 0.2 mg/ml of 5-bromo-4-chloro-3-indoyl phosphate in 10 ml of 0.1 M Tris/HCl, pH 9.5,0.1 M NaCl and 10 mM MgC12 for up to 5 h. The reaction was stopped with 10 mM Tris/HCl, pH 7.5, and 10 mM EDTA.

Results

Sensitivity and specificity of PCR

For determination of the sensitivity of PCR, lo-fold dilutions of purified HCMV (Towne strain) DNA and culture supernatant from infected cell culture were subjected to PCR. By direct gel analysis, 0.01-0.3 pg of HCMV DNA, corresponding to 30-100 molecules of HCMV DNA molecules and l-5 plaque- forming units (PFU) could be detected. The sensitivity increased more than lo- fold by Southern blot hybridization with nonradioactive oligonucleotide probes, which indicates that a few copies of HCMV DNA molecules are detectable. The sensitivities obtained by using two pairs of primers MIE and pp71 were at almost the same levels. In addition, very few cases were found to be false negative when pp71 primers were used. Therefore, the data obtained mainly by the use of MIE primers were shown here.

The following samples were used as template DNAs for the evaluation of the specificity of PCR: DNA (10 ng) extracted from HEL cells infected with herpes simplex virus type 1 (HSV- 1, HF strain) and herpes simplex virus type 2 (HSV- 2, UW265 strain) at the late stage of their growth cycles; DNA (10 ng)

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extracted from mock-infected HEL cells, Epstein-Barr virus (EBV)-producer B95 cells; DNA (10 pg) purified from virions of varicella-zoster virus (H-N3 strain, provided by Dr. R. Hondo, Institute of Medical Science, Tokyo University). Specific amplification was not detected by direct gel analysis and confirmed by Southern blot hybridization. Furthermore, all DNAs extracted from cells infected with 13 HCMV isolates showed amplified products with the same size as expected from the target sequences for two sets of primers used here, which indicates that the sequences were well conserved in HCMV DNAs from various isolates.

Detection of HCMV DNA in urine samples

Figure 1 shows examples of PCR determined by direct gel analysis stained with ethidium bromide and Southern blot hybridization with a nonradioactive oligonucleotide probe linked with alkaline phosphatase and Table 1 summarises the results obtained by use of 19 urine specimens Ul to U19.

A

B

C

D

IJIO U2 u15 Sample M PC NC

/hn\ 30 5 1 0.1 30 5 1 0.1 30 5 1 0.1 Urine Vol.( K 1)

426bp

- 426bp

u7 U8 U18 U18+HCMV Sample

30 5 1 0.1 30 5 1 0.1 30 5 1 0.1 30 5 1 0.1 0 Urine V0l.t I I)

126bp

Fig. 1. Detection of HCMV in various amounts of urine by PCR using MIE primers. Urine samples with (A and B) or without (C and D) infectious HCMV were subjected to PCR and the amplified products were examined by direct gel analysis (A and C) and Southern blot hybridization (B and D). Symbols: M, DNA molecular weight marker pHY (Takara Shuzo Co., Kyoto); PC, positive control, DNA (10 ng) extracted from cells infected with Towne strain; NC, negative control, DNA (10 ng) extracted from uninfected MRC-5 cells. U18 + HCMV indicates the sample including U18 and the culture fluid with HCMV of 2.5 PFU. The number in bp on the left of A shows the fragment sizes of the DNA molecular weight marker.

Bands of 426 bp show detection of products amplified by MIE primers.

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TABLE 1

Detection of HCMV in urine samples by tissue culture and PCR

Sample culture Band intensity of amplified product

Urine volume

30 PI 5 pl 1 Id 0.1 jll I

DC SB DG SB DG SB DG SB

UlO u2

:;7 U16 u15 Ul,U5 u7 U6,U9

:;t Ull, u13 u12, u14 Ul8, u19

+ + + + + + + + + - _ + + + - _ - + + _ + + - - - -

- - _ - + - - _ - +

+ + + + + + - + _ + _ + - + _ + _ + - * _ f _ + _ +

+ + + + _ + _+ + - + _ + + + * + f + * + - + _ + - +

Culture: + , HCMV positive; -, HCMV negative. Band intensity: + , visible; &, slightly visible; -, not detectable. DG, direct gel analysis; SB, Southern blot hybridization.

Except for one sample UlO (Fig. lA), all samples examined were negative by both direct gel analysis and Southern blot hybridization when 30 ~1 of urine samples were included in 50 ~1 of the PCR reaction mixture. Among the urine samples which had been shown to contain infectious HCMV by tissue culture, U2 was positive by direct gel analysis when the sample volume decreased to 5 ~1

A Culture + +

Sample UIO u2 u4 NC M PC

Urine Vol.( ~1) 30 5 i 0.1 30 5 1 0.1 30 5 1 0.1

Fig. 2. Detection of HCMV in urine by PCR using pp71 primers. (A) Direct gel analysis of amplified products. (B) Southern blot hybridization of A. Symbols M, PC and NC are as described in Fig. 1. Bands

of 316 bp show detection of products amplified by pp71 primers.

- 316bp

- 316bp

215

U18 - U18 U18 u13 u4 U18 ---

-f-f- -l- - - __

_ + +- +- f- - - -

1 3 6 16 16 16 16

Urine Sample

HCMV

PEG Treatment

Incubation Time(h)

- 426bp

Fig. 3. PCR analysis of PEG-treated urine. MIE primers were used for amplification of HCMV DNA. Urine samples were incubated with PEG for the time period as indicated. Direct gel analysis was shown

here. HCMV indicates the culture fluid containing HCMV of 15 PFU which was added to U18.

and U15 was negative by direct gel analysis even when decreased to 0.1 ~1, indicating the presence of PCR inhibitors in these samples (Fig. 1A). However, all these samples were positive by Southern blot hybridization when 0.1 or 1 ,LL~ was used (Fig. 1B). Fig. 1C and D show the results of urine samples which were negative by virus isolation in culture. Of these samples, U7 and U8 in 1 ~1 and 0.1 ~1, respectively, were slightly positive even by direct gel analysis. As shown in Table 1, a significant number of urine samples that were negative by virus isolation gave positive results by PCR followed by Southern blot hybridization. U18 and U19 were negative by both direct gel analysis and Southern blot hybridization.

We also examined the inhibitory effect on the detection of HCMV by PCR using various volumes of U18 and the HCMV culture fluid of 2.5 PFU (Fig. 1C and D). HCMV with 2.5 PFU was detected in the absence of Ul8 even by direct gel analysis whereas addition of more than 5 ~1 of U18 to HCMV with 2.5 PFU resulted in no amplified product, which indicates that U18 contained PCR inhibitors.

Figure 2A and B show examples of specific amplification by use of pp71 primers. These findings indicate that the hybridization procedure using a nonradioactive probe increased the sensitivity for PCR and is useful for detection of HCMV in urine by PCR.

Precipitation of HCMVfrom urine by PEG

To remove PCR inhibitors from urine, extensive purification of DNA by standard extraction procedure using phenol may be best. However, more practical methods are required for routine tests. First, we attempted to remove PCR inhibitors from U18 by precipitation of HCMV with PEG using the HCMV-infected culture fluid of 15 PFU. The mixture was treated with PEG

216

U18 - U18 u13 u4

- + ++ - -

Urine Sample

HCMV

G P Treatment

- 426bp

Fig. 4. PCR analysis of DNA extracted from urine with glass powder (GP). MIE primers were used for amplification of HCMV DNA. Direct gel analysis was shown here. HCMV as described in Fig. 3.

6000 for various periods on ice and subjected to PCR. After PEG treatment, the amplified product of U18 with 15 PFU, HCMV was clearly visible even at an incubation time of 1 h by direct gel analysis while PEG-treated U18 without 15 PFU HCMV gave a negative result (Fig. 3). The longer incubation time appeared to yield more amplified products. The amplified product was not derived from PEG-treated U18 because the PCR product of PEG-treated U18 in 30 ~1 was not detected even by Southern blot hybridization (data not shown). When PEG-treated samples U13 and U4 from each 30 ~1 containing no infectious HCMV were subjected to PCR, the amplified products were found to be clearly visible by direct gel analysis (Fig. 3). Specific amplification was also obtained when PEG 1000 and 50 000 were tested at final concentrations of 1, 8 and 16% and when PEG 200 000 was at a final concentration of 0.4% (data not shown).

Extraction of HCMV DNA with glass powder from urine

As the second method of removing PCR inhibitors from urine, the glass powder procedure was tested by PCR using a mixture of U18 in 30 ~1 with HCMV of 15 PFU. The amplified products of DNAs extracted from the mixture with glass powder were clearly positive by direct gel analysis (Fig. 4). Extraction from urine samples containing no infectious HCMV such as U13 and U4 resulted in specific amplification as clearly visible even by direct gel analysis. As shown by PEG treatment (Fig. 3), no specific amplification was observed in the DNA extracted from 30 ~1 of U18 with glass powder. Therefore, the use of glass powder increased the sensitivity of PCR by removal of the inhibitors as found in PEG-treated urine samples.

217

Discussion

HCMV DNA can be detected in 0.1-5 ~1 of urine samples by PCR without purification of viral DNA as reported by Demmler et al. (1988). However, it was found that many urine samples containing infectious HCMV showed no specific or little amplification by direct gel analysis of PCR products, due to inhibitors for PCR. For increased sensitivity of PCR, we employed Southern blot hybridization with nonradioactive oligonucleotide probes which could detect as little as 1 pg of HCMV DNA. The use of the Southern blot hybridization revealed that all samples containing infectious HCMV examined were positive for specific amplification. Apart from the failure of HCMV isolation by tissue culture, PCR of many urine samples such as U4 and U13 showed specific amplification when the amplified products were examined by Southern blot hybridization. Under the conditions used here, specific amplification was not observed in the negative controls. Thus, Southern blot hybridization used here is useful for PCR of urine samples since the use of a nonradioactive probe is essential in the clinical laboratory, especially to prove the specificity of PCR. For preliminary screening of amplified products, it is recommended that dot-blot hybridization is used with the nonradioactive oligonucleotide probes described here. The use of dot-blot hybridization can save the steps for electrophoresis and Southern transfer of amplified products.

PCR inhibitors were removed at least partially from urine by either PEG treatment or the glass powder method. All urine samples examined which were negative by direct gel analysis but positive by Southern blot hybridization were clearly visible by direct gel analysis after these treatments. These two methods appeared to be equally effective for removal of PCR inhibitors from urine. However, the total time required for the DNA extraction process by the glass powder method was about 1 h, being slightly shorter than PEG treatment which required at least 2 h. Kimpton et al. (1990) incubated urine samples with PEG overnight, probably because they needed to precipitate enough viruses for detection of dot-blot hybridization. In addition, Landini et al. (1990) showed that PEG treatment was not satisfactory for detection of HCMV in PEG- treated urine by dot-blot hybridization because the thick PEG precipitate directly dotted onto the filter could be released. The use of PCR under the conditions described here did not include the step for direct blotting of PEG- treated samples and increased the sensitivity, probably by about lOO-fold in comparison with previous studies using dot-blot hybridization of PEG-treated samples (Kimpton et al., 1990; Landini et al., 1990). In addition to 19 urine samples examined here, we have tested more than 30 clinical samples which were not examined for the presence of infectious HCMV by tissue culture. Most of these samples showed that the amplified products were detected only by Southern blot hybridization or slightly by direct gel analysis.

It is necessary to fix the sample volume for clinical tests with a large number of samples. Therefore, it is better to treat urine samples with PEG or glass powder prior to PCR and for the detection of the amplified products by direct

218

gel analysis. Otherwise, urine in varied volumes must be tested for each sample by PCR as shown by Demmler et al. (1988) and in Table 1. This is also one of the novel points of this work.

Thus, both PEG treatment and the glass powder method are useful for application of many samples at any one time and can be carried out easily in ordinary clinical laboratories.

Acknowledgements

The authors wish to thank Drs. Y. Tsutsui and R. Hondo for providing samples.

This work was supported by grants from the Ministry of Education, Science and Culture, Japan.

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