In situ hybridization detection of short viral amplicon sequences within cultured cells and body...

Journal of Virological Methods

ELSEVIER Journal of Virological Methods 52 (1~95) 247-263

In situ hybridization detection of short viral amplicon sequences within cultured cells and body

fluids after the in situ polymerase chain reaction

Ronnie Ray a3b, Mark Smith b, Rosalind Sim b, Ian Bruce ‘, Andrew Wakefield by *

a Department of Pathology and Laboratory of Molecular Biology, Tufts University School of Medicine,

New England Medical Center Hospital, 750 Washington Street, Boston MA 02111, USA

b Inflammatory Bowel Disease Study Group, Royal Free Hospital, Pond Street, Hampstead,

London NW3 2QG, UK

’ Department of Molecular Biology, Uniuersity of Greenwich, Wellington Street, Woolwich,

London SE18 6PF, UK

Accepted 27 July 1994

Abstract

Using single primer pairs, intracellular gene sequences of cytomegalovirus (CMV-Towne’s strain) and cY-tubulin were amplified (in situ PCR) from cells in human body fluids and in suspensions. Visualization of CMV amplificants was carried out by in situ hybridization (ISH), using both a biotinylated double-stranded DNA probe and a radiolabelled oligonucleotide probe. Visualization of cY-tubulin amplificants was achieved using both radiolabelled single-stranded cRNA and oligonucleotide probes. Liberated amplificants were also identified by bands of

expected size by gel electrophoresis. The specificity of the PCR products was confirmed by Southern blot analysis. Intracellular amplification was identified both in unfixed cells and, optimally, after brief alcohol fixation, whilst maintaining relative isotonicity in all working solutions. For CMV, enhanced signal was observed in cells (cultured fibroblasts or urine sediment) undergoing in situ PCR using either biotinylated or radiolabelled probes compared with controls undergoing ISH alone. For cy-tubulin, radiolabelled riboprobes and oligoprobes only produced signals within cells (human peripheral lymphocytes, ascitic fluid and bladder washings from routine cytological specimens) after in situ PCR, but not after ISH alone. Morphological evaluation was superior with biotinylated probes, and minimal back-diffusion effect was found compared with radiolabelled probes. Up to 80% of cells survived thermal cycling. In situ PCR

* Corresponding author

0166-0934/9.5/$09.50 0 1995 Elsevier Science B.V. All rights reserved

SSDI 0166-0934(94)00117-O

248 R. Ray et al. /Journal of Virological Methods 52 (I 995) 247-263

detected short sequence (100 bp) foreign DNA and low copy number genomic DNA, and was superior to ISH alone. In contrast to radiolabelled probes, very small CMV amplificants could be detected without a significant ‘back-diffusion’ effect when using the large biotinylated probe in this model system.

Keywords: In situ PCR; In situ hybridization; Polymerase chain reaction; Cytomegalovirus

1. Introduction

The purpose of the present study was to test the feasibility of performing the intracellular polymerase chain reaction (in situ PCR) for both foreign (CMV) and human genomic sequences within body fluids and cultured cells, without protease pretreatment

steps that might compromise morphology. Since cytological fixatives are largely alcohol-based, we examined the effects of both brief alcohol fixation with either no fixation, or with other fixatives. In addition, we wished to test the feasibility of, and potential

artifacts arising from, detection of very short amplificant sequences by non-isotopic

probes, and to examine possible differences between radiolabelled and non-isotopic probes for in situ hybridization (ISH) detection of amplified products. In order to

optimize morphology, relatively isoosmotic (isotonic) working solutions were used for

the study. Haase et al. (1990) have demonstrated in situ PCR amplification of visna virus

genome in cultured sheep cells using multiple overlapping primers (multiplex PCR) with subsequent visualization by ISH with ‘virus-specific’ DNA sequences. The production of long amplificants generated by overlapping PCR products was designed to minimize

leakage from cells. Subsequent work has shown the feasibility of in situ PCR using single primer pairs (Ray et al., 1991). More recently, Bagasra et al. (1992) and Nuovo

and colleagues (1991, 1992) have used single primer pairs for in situ PCR followed by ISH detection of genetic sequences within cells (Fig. 1). A number of potential problems

Primers Jaq Polymerase

Nucleotides

In situ hybridisation In situ ampliiication

Fig. 1. In situ detection of genomic sequences.

R. Ray et al. /Journal of Virological Methods 52 (1995) 247-263 249

prevent further progress towards use of this technology for both routine cytological diagnostics and research; these include the presence, in particular, of inhibitors of the PCR reaction in body fluids and the ‘back-diffusion’ effect of extracellularly formed

amplificants into cells.

The presence of inhibitors of PCR on extracted DNA are well known, and have been

reported specifically, for detection of CMV sequences in urine (Khan et al., 1991). It is

possible that by maintaining membrane integrity during the in situ PCR reaction, these

inhibitors may be prevented from traversing membranes, thereby permitting the in situ

PCR reaction to progress unhindered.

‘Back-diffusion’ of small amplificants (196 bp) into cells has been documented

previously, using small radioactive oligonucleotide probes (Komminoth et al., 1992).

However, addition of non-isotopic labels into amplificants significantly reduced this

‘back-diffusion’ effect when detection was carried out with the same radioactive probes.

This may have been due to either a size or charge effect of the non-isotopic label which

may have prevented extracellularly formed amplificants from back-diffusing across

membranes, or alternatively, sticking to them. A small uncharged probe bound to a very

small target (100 bp) might traverse membranes with ease: this may be less likely if the

small target is bound to a very bulky and highly charged probe. In this way hybridized extracellular target/probe complexes may be trapped within cells and withstand wash-

ing steps with relative ease. The purpose of this study was to develop a protocol for in situ PCR for cytological

analysis, that overcomes many of the problems encountered previously.

2. Materials and methods

2.1. Oligonucleotide primers and probes

The genes studied included cytomegalovirus (CMV) Towne strain infected cells (Darlington et al., 1991; Ray et al., 1991; Komminoth et al., 1992) and the human

cu-tubulin gene family (Ray et al., 1991; Komminoth et al., 1992). The primers were tested initially on extracted genomic DNA and the specificity of amplification was documented both by agarose gel band size analysis and by Southern blotting. Oligonu- cleotides used as primers for PCR, and probes for Southern blot or ISH (Fig. 2) were

synthesized using a DNA synthesizer (Model 380, Applied Biosystems, Foster City, CA) purified, dried, resuspended in TE buffer (Tris EDTA buffer pH 8.0) and quantified by spectrophotometer measurement at OD 260 nm (Spectronic 1001, Bausch and Lomb) then stored at 4°C.

2.2. Cells

A human fibroblast cell line MRC-5 (Whittaker MA Bioproducts, Walkersville, MD, USA) infected (or uninfected) with either CMV or herpes simplex virus (HSV) II, Vero cells (green monkey kidney) infected (or uninfected) with measles virus (Edmonston strain) and rat adrenal cells, were cultivated in RPM1 1640 with Eagle’s minimal

250 R. Ray et al. /Journal of Virological Methods 52 (1995) 247-263

SYNTHETIC OLIGONUCLEOTIDES USED AS PCR PRIMERS

GENE

CMV (glycoprotein B) (Courtesy of Dr J Fox, University College and Middlesex School of Medicine, LONDON UK).

CMV (major intermediate early)

NUCLEOTIDE LOCATION

1967-1989 2066-2044

731.755 1165-1150

SEQUENCE PRODUCT SIZE (BASE PAIRS)

5’ ACC ACC GCA CTG AGG AAT GTC AG 100 3’ 5’ TCA ATC ATG CGT TTG AAG AGG TA 3’

5’ CCA AGC GGC CTC TGA TAA CCA 435 AGC C 3’ 5’ CAG CAC CAT CCT CCT CCT CCT Cl-G G 3’

Human alpha-tubulm 402.421 (sense) 5’ CCT C(yT GGT TIT CCA CAG CT 3’ 640 1041-1022 5’ GCA CCA ATC CAC AAA 0-G GA 3’ (antisense)

SYNTHETIC NUCLEOTIDES USED AS PROBES FOR SOUTHERN BLOT AND ISH

Fig. 2. Synthetic oligonucleotides used as PCR primers and probes.

essential medium, 1% glutamine, 2% fetal bovine serum, penicillin G (200 U/l ml), streptomycin (200 pg/ ml) and amphotericin B (0.5 pg/ ml). The cells were stored at -70°C and thawed immediately before use. Aliquots from clinical samples of ascitic

fluid, bladder washings, and CMV-infected and uninfected urine sediment, were also

studied. Peripheral blood B-lymphocytes were separated from anticoagulated venous blood using a Ficoll-Hypaque density gradient centrifugation (Pharmacia, Piscataway,

NJ, USA), followed by sheep erythrocyte rosetting and plastic adherence. Cells were counted before and after PCR in a haemocytometer (Buffalo, NY, USA)

or Sysmex NE8000 (Baxter Healthcare Corp, McGaw Park, IL, USA). Cells, serially

diluted in phosphate-buffered saline (PBS), were used to evaluate the minimum number of cells required for in situ PCR. The osmolarity of the PCR mixture and other working

solutions was measured using an osmometer (Model 3M0, Advanced Instruments Inc, Needham Heights, MA, USA). Cell samples were adjusted to concentrations of 50,000- 100,000 cells/ml, and 400 ~1 from each sample was aliquoted into 0.5 ml Eppendorf tubes and centrifuged at 1500 rpm for 2 min. Cells were resuspended in 50 ~1 of

fixative and incubated for 3-15 min at room temperature. The fixatives used were 10% buffered formalin, Bouin’s solution, Trump’s solution and lo-100% alcohol or no

fixation. Cells were washed in PBS prior to being resuspended in the PCR buffer. In some cases, proteinase K predigestion (1 mg/ml for 15 min) was performed.

2.3. In situ PCR

Centrifugation was carried out at 1500 rpm for 2 min and cells were gently

resuspended in 178 ~1 PCR mixture comprising 5 ~1 2.5 mM of each dNTP, 20 ,ul


Fixation (95% ETOH)

I

Centrifugalion

Washing (PBS)

1

Centrifugalion

Polymerase Chain Reaction

/\ Cell lysis Cytospins

t t Gel Electrophoresis In-situ Hybridization

(Southern Blot)

Fig. 3. In situ PCR procedure. Detection of amplification by independent techniques.

10 X PCR buffer, (100 mM Tris HCl at pH 8.3, 500 mM potassium chloride, 25 mM

magnesium chloride and 1 mg/ml bovine serum albumin), 134 ml distilled water and 2

~1 of each relevant primer pair (lo-50 pmol). Next, 0.5 ~1 (5 U/pi) Thermus aquaticus Tuq polymerase (Perkin-Elmer/Cetus, Norwalk, CT, USA) was added followed by two drops of mineral oil. A range of thermal cycling parameters for in situ

PCR were studied. The following specifications were found to be optimal for the primer pairs used. Denaturation, 94”C, 1 min; reannealing, 58”C, 2 min; extension, 74”C, 1.5

min with a 5-s extension added to each cycle, 40 cycles (Fig. 3). A 20-~1 aliquot from each tube after PCR was freeze-thawed 3 times to lyse cells and release intracellular PCR products, then centrifuged at 5000 rpm to concentrate cell debris into a pellet.

Twelve microlitres of the supernatant of each sample was removed and mixed with 4 ~1 DNA-loading dye, and electrophoresed with ethidium bromide (10 mg/ml). Molecular weight markers (PUC 19 digested with Dde 1) were included with each gel, and results

recorded on Polaroid photographs using ultraviolet fluorescence. The remaining 158 ~1

of each post-PCR sample was cytospun onto plain or glued (salinized or gelatinized) slides (1000 rpm for 2 min) using a Cytospin-Centrifuge (Shandon Inc., Pittsburg, PA,

USA) air-dried, baked overnight at 42°C and then stored at room temperature. Finally, in situ hybridization was performed as described below, and the results

evaluated by blinded studies.

2.4. In situ hybridization

2.4.1. ISH with biotinylated CMV DNA probes ISH using virus-specific biotinylated probes (Enzo Diagnostics, Inc, NY, USA) was

performed using a one-step detection procedure according to previously described

252 R. Ray et al. /Journal of Viirological Methods 52 (1995) 247-263

methods (Pardue and Gall, 1975; Brigati et al., 1983). ISH experiments were carried out

with and without prior proteinase K (1 mg/ml for 15 min at 37°C) incubation.

2.4.2. ISH with 3sS-labelled cu-tub&n riboprobes

Generation of human cY-tubulin antisense mRNA probes and the ISH procedure were

carried out according to the method described (Hoefler et al., 1986) with an additional

denaturation step before hybridization, which consisted of heating the slides to 95°C on a hot plate and then cooling on ice for an additional 5 min.

2.4.3. ISH with 3sS-labelled CMV and cx-tubulin oligoprobes

ISH was performed according to the method of (Long et al., 1992) using probes as

shown (Fig. 2).

2.5. Southern blotting of PCR products

The specificity of PCR-amplified DNA fragments was evaluated by Southern blotting

using relevant ‘kinased’ oligonucleotide probes (Sambrook et al., 1989).

2.6. Nested polymerase chain reaction for CAN

Nested PCR for glycoprotein B of CMV was carried out using an outer flanking primer pair and inner flanking primer pair (Fig. 2) as described previously (Darlington et

al., 1991).

2.7. Specificity controls

The following were employed as specificity controls for the combined in situ PCR

and ISH experiment: (1) cells not subjected to in situ PCR; (2) cells soaked in the PCR mixture but not subjected to thermal cycling; (3) cells subjected to thermal cycling

without Taq polymerase in the PCR mixture; and (4) cells subjected to thermal cycling without primers in the PCR mixture. As controls for hybridization with internal oligonucleotides in both Southern blot and ISH, irrelevant oligoprobes (either CMV

probe for a-tubulin amplificants or cw-tubulin probe for CMV amplificants) were used. A control riboprobe with specificity for c-myc was used in ISH experiments using the cY-tubulin-specific riboprobe. In all experiments, the following negative control samples were used: HSV-II-infected or uninfected cells, for CMV, and mouse adrenal cells for human cY-tubulin in body fluids, fibroblasts and lymphocytes.

2.8. Back-difision

It was possible that large amounts of amplificants produced from DNA liberated

during thermal cycling may have ‘back-diffused’ into cells or adhered to membrane

surfaces. To test this theoretical possibility, in 6 experiments alcohol-fixed HSV-II-infected fibroblast cells, measles-infected Vero cells, or human lymphocytes (50,000- 100,000/ml) were suspended in an isotonic PCR mix containing preformed 100 base


pair CMV amplificants, liberated from aliquots of CMV-infected fibroblasts which had

been lysed after in situ PCR.

2.9. Relative ejjiciency of reaction

In order to compare the relative efficiency of in situ PCR with amplification of

extracellular DNA (CMV, cy-tubulin), cell suspensions submitted for in situ PCR were centrifuged and divided into supernatant (extracellular amplificants) and cell (in situ

PCR amplificants) fractions. The cell fraction was then washed in PBS, lysed by freeze

thawing and resuspended in PBS to an identical volume as the supematant fraction. The intensities of the amplificant bands in both supematants and cell fractions (resuspended

in an identical volume of phosphate-buffered saline) were then compared by gel

electrophoresis.

3. Results

3.1. Cell preservation

It was found that isotonicity in the PCR mixture and other working solutions was

helpful in preserving morphological characteristics during thermal cycling. Despite the potential for cell injury and loss due to thermal cycling, fixation, washing and centrifu-

gation steps, up to 80% of cells consistently survived the procedure. Centrifugation rates above 400 g after fixation resulted in cell clumping and loss; however, body fluids were more robust (50% survival) than cultured fibroblasts (20% survival) at 400 g.

Cellular morphology was moderately well preserved even after 60 cycles, particularly

for human peripheral lymphocytes and the human fibroblasts cell line (MRC-5). However, proteinase K pretreatment resulted in cell destruction at concentrations used.

3.2. Gel electrophoresis and southern blot

After testing primer pairs for CMV and a-tubulin (Fig. 2) with appropriate genomic

DNA by solution phase PCR, identical primer pairs were used for in situ PCR reactions: corresponding amplificants liberated after cell lysis revealed bands of expected size by gel electrophoresis (Fig. 4). Southern blot analysis with radiolabelled oligonucleotide probes internal to the primers, showed specific hybridization for respective PCR

products (Fig. 4). Nested PCR reactions produced a 100-bp product by gel electrophoresis using primers previously described for glycoprotein B in CMV (Darlington et al., 1991); cells lysed after single round in situ PCR, using inner flanking primers alone, liberated an identical PCR product.

As shown using alcohol-fixed CMV-infected fibroblasts as template for CMV

primers, cells lysed after in situ PCR liberated amplificants of expected size that were absent in negative control samples (Fig. 5). Not all fixatives were equally permissive for in situ PCR, since amplification was detectable only in unfixed cells and after 95% alcohol fixation (Fig. 6). PCR products were not seen in cells fixed with Bouin’s

254 R. Ray et al. /Journal of Virological Methods 52 I1 995) 247-263

235

426 540

910

a b

VH CMV AT NT VH CMV AT NT Fig. 4. (a) Specificity of amplified products liberated by cell lysis demonstrated by gel electrophoresis. (b)

Southern blot analysis. Cytomegalovirus (CMV), cu-tubulin (AT), VH3 gene rearrangement (VH) and

neurotensin (NT), were used as controls to confirm successful Southern blot procedure.

solution, Trump’s solution or 10% buffered formalin. Investigation of different concentrations of alcohol fixation revealed excellent in situ PCR amplification using SO-100%

alcohol; below 50%, amplification decreased progressively as the alcohol content fell, although unfixed samples also showed amplificant bands. Alcohol was the best fixative for the sequences investigated: length of fixation (3-15 min) was not critical to the in

situ PCR reaction for these sequences. Pretreatment of cells ,.vith proteinase K prior to in situ PCR resulted in strong bands

in the gel analysis of an intensity equal to that found without a proteinase step; however,

morphology was severely compromised compared with undigested cell preparations.

cells g&JPCR

a. CMV + +

b. None + +

C. CMV+ + -

d. CMV+ - -

e. HSVII+ + +

f. HSVII+ + -

6. HSVII+ - - size marker

Fig. 5. Amplificant band liberated from alcohol-fixed CMV-infected fibroblasts lysed after in situ PCR (with

appropriate controls), demonstrated by gel electrophoresis.

R. Ray et al. /Journal of Virological Method.7 52 (1995) 247-263 255

235

426 540 910

Fig. 6. Effect of different fixatives on CMV-infected fibroblasts. 3 min fixation or incubation before in situ

PCR. No predigestion. Amplificant band liberated from cells lysed after in situ PCR; demonstrated by gel

electrophoresis. F, formalin; A, 95% alcohol; B, Bouin’s solution; T. Trump’s solution; N, no fixative.

When comparing the relative efficiency of extracellular and intracellular amplifica-

tion by gel electrophoresis after 40 cycles, appropriately sized bands were noted for each

respective gene sequence (CMV and human a-tubulin) in both supernatant (extracellular amplificant) fraction and cellular (in situ PCR) fraction diluted to the same volume.

CMVS r

ATUB’ r

Fig. 7. Comparison of relative efficiency of PCR in supernatant (s) and cellular residue (r) fractions.

256 R. Ray et al. /Journal of Vimlogical Methods 52 (19951 247-263

However, the band intensity was greater in the supernatant fraction, consistent with a greater efficiency of PCR on liberated DNA compared with in situ PCR (Fig. 7).

All cell samples not undergoing intracellular amplification showed no bands on gel

electrophoresis. HSV-II-infected cells showed no PCR product after in situ PCR was

performed using CMV-specific primers. Contamination with extraneous DNA was excluded by failure to detect bands by gel electrophoresis on PCR samples from which

either cells, or DNA template were omitted.

No cross-hybridization of oligoprobes was found in Southern blot experiments.

3.3. Visualization of PCR products by ZSH

A biotinylated DNA probe specific for CMV was used for ISH in 10 experiments on

CMV-infected fibroblasts which had been subjected to in situ PCR amplification, and

compared with similar cells not undergoing in situ PCR. An intense intranuclear ‘block-like’ staining pattern was observed, when compared with ‘grain-like’ staining of

unamplified samples (Fig. 8). Similar results were found for CMV-infected urinary

sediment. HSV-II-infected fibroblasts showed no signal. A radiolabelled single-stranded cRNA probe was employed for ISH in 8 experiments

to detected amplified cy-tubulin. A strongly positive result was seen after 5 days’ exposure (Fig. 9) for human peripheral lymphocytes, ascitic fluid and bladder washings;

however, radioactive ‘grains’ tended to obscure underlying morphology. No increased signal relative to background was found for unamplified samples, when a c-myc-specific

riboprobe was used for detection, or when in situ PCR was performed on mouse adrenal cells using a-tubulin-specific primers.

Oligonucleotide probes were used for ISH in 8 experiments after in situ PCR. The amplified sequences comprised CMV (4 cases) and a-tubulin (4 cases). In all cases, large numbers of nuclear and perinuclear autoradiographic grains were seen intracellu-

larly after 2-5 days’ exposure. Cells that were studied included human peripheral

lymphocytes, ascitic fluid, bladder washings (a-tubulin) and a fibroblast cell line (CMV). Radioactive ‘grains’ tended to obscure underlying morphology. The background was very low with all ISH probes, giving strong signal-to-background ratios. This was

achieved without postfixation. For CMV-infected cells, unamplified samples showed a signal which was not as intense as that found for amplified cells. However, unamplified cells showed no increased signal relative to background when ISH was carried out with the tubulin-specific oligoprobe. Cross-hybridization of oligoprobes was not found. NO

cellular signal was found when in situ PCR was performed either on mouse adrenal cells using cr-tubulin-specific primers, or on HSV-II-infected fibroblasts using CMV-specific

primers.

3.4. ’ Back-difision ’ effect

Similar to the findings of others (Komminoth et al., 1992) using radiolabelled oligoprobe ISH for CMV, positivity was noted when HSV-II-infected fibroblasts and human lymphocytes were bathed in a PCR mix containing preformed CMV amplificants (435 bp) as described above. This effect was more intense at 40 cycles than at 30 cycles.

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The pattern of the grains tended to assume a ‘ring-like’ form around the cells and to be

predominantly peripheral cytoplasmic, as compared with the mainly nuclear distribution found for in situ PCR (Fig. lOa). No increased signal relative to background was

detected using the biotinylated CMV-DNA probe when either HSV-II-infected fibrob-

lasts or measles-infected Vero cells were bathed in a PCR mixture containing preformed

CMV amplificants (435 or 100 bp) and 40 cycles of thermal cycling performed (Fig. lob). This was in sharp contrast with the distinct ‘block-like’ nuclear and cytoplasmic

staining pattern for in situ PCR on CMV-infected fibroblasts, and the ‘grain-like’ nuclear and cytoplasmic staining pattern for unamplified CMV-infected cells, using the

same biotinylated CMV probe. The findings are listed below.

(1) High alcohol concentrations without protease predigestion are suitable for in situ . PCR experiments (fixation time 3-15 min); progressively diluted alcohol concentra-

tion produces less amplification. No postfixation is necessary. (2) There is correlation between cycling conditions for in situ PCR and PCR on

extracted DNA for the sequences tested.

(3) In situ PCR is suitable for the body fluids despite the presence of inhibitors of reaction. Fifty percent of body fluids survive reaction compared to 20% of viral-in-

fected cultured cells. (4) Up to 60 cycles of in situ PCR may be performed with some preservation of

morphology. (5) Isotonic solutions are highly suitable for in situ PCR reaction. (6) ‘Back-diffusion effect’ of very small amplification (100 bp) is ameliorated by large

(and charged) non-isotope probes during the ISH step. (7) Radioactive ‘grains’ obscure underlying morphology. (8) Extracellular amplification is much more efficient than intracellular amplification.

4. Discussion

A method if described for in situ PCR which can be used for the amplification of both viral gene sequences (CMV) (Kinney et al., 1985; Loning et al., 1986; Demmier et al., 1988; Stock1 et al., 1988; Cassol et al., 1989; Olive et al., 1989; Dankner et al.,

1990) and previously characterised sequences of human a-tubulin (Cleveland et al., 1980; Lemischka et al., 1981; Cowan et al., 1983; Hall and Cowan 1985) in different cytological samples and cultured cells. By combining intracellular amplification by PCR

with the specificity and morphological preservation of ISH using a simple, reproducible and relatively cheap method, we believe that this technique has great practical value for research, and for routine cytological and pathological diagnostic practice. Both CMV-infected fibroblasts and urine sediment have multiple viral genomic copies per cell: in contrast, the human a-tubulin gene, which codes for cytoskeletal proteins, is a gene

family with several gene copies per cell (Cleveland et al., 1980). In this study, alcohol fixation of cells prior to in situ PCR gave excellent amplifica-

tion results. This is of interest since many cytological fixatives are alcohol-based. Alcohol fixation may cause mild disruption of cellular lipid membranes (Sheehan and Hrapch, 1973); in addition, it may precipitate membrane protein to an extent that permits the components of the PCR reaction to diffuse into the cells, but apparently prevents


amplificants from escaping, whilst preventing leakage of nucleic acid degrading en-

zymes from lysosomes. Lack of amplification with 10% buffered formalin, Bouin’s or Trump’s fixation may be due to difficulty in removing residual fixatives in washing

steps, resulting in inhibition of Taq polymerase enzyme. It is possible that the failure of amplification after fixation in 10% buffered formalin may also relate to crosslinking of

protein in the cell membrane, cytoskeleton and nucleus (Sheehan and Hrapch, 1973;

Junqueira et al., 1977), thereby reducing the accessibility of the DNA template. Bouin’s fixative appeared to eliminate the primer blush on gel electrophoresis, suggesting

destruction of primers. However, Nuovo and colleagues (1991 and 1992) found 10% buffered formalin to be an excellent fixative after protease treatment, which may release

crosslinked DNA-binding histones and cell membrane proteins, thus permitting access of

PCR reaction components. Proteinase K, which digests proteins, did not inhibit gene

sequence amplification as shown by gel electrophoresis, but did result in significant cell lysis after alcohol fixation. A post-fixation step was not found necessary for reaction. Of particular interest, cells which were unfixed yielded amplificant bands, although these

were less intense than those of alcohol-fixed cells. This may be due either to small

amounts of Tween in commercial PCR reagents, mild perturbations in cell membranes produced by viral infection, or to membrane disruption in thermal cycling. In contrast

with well-dispersed cells, those cells at the centre of large cell clumps tended to show a decreased signal, suggesting poor penetration of probe (or visualizing antibody) within clumps. A further point of interest is that maintaining isotonicity throughout the

procedures may reduce osmotic pressure effects on cells, lessen problems of evaporation and reduce the use of expensive PCR mixture components. Finally, isotonic (and therefore diluted) solutions may be relatively specific compared to concentrated counter-

parts (Innis and Gelfand, 1990); since some evaporation is inevitable at the end of reaction, reaction components concentrate in proportion, thereby counteracting effects of deterioration in Tuq polymerase enzyme towards the end of cycling.

The same thermal cycling conditions for a particular primer pair on extracted DNA can be used with in situ PCR reactions. In accordance with the findings of Haase et al. (1990) in situ PCR is less efficient than PCR on purified template and may require more

cycles to achieve a similar degree of amplification. However, it was observed that when fixed cells were used as template, 40 cycles gave intense amplification, but larger cycle numbers yielded less product, perhaps due to loss of PCR substrate components and

permanent denaturation of DNA structure by excessive cycling. No evidence of non- specific amplification was found in our study, and so ‘hot-start’ methods were not required. ‘Hot-start’ also suffers from potential problems of starting reactions simultane-

ously for all samples, contamination problems, difficulty in mixing reagents: further- more, hot-start cannot by itself eliminate problems of mispriming (Long et al., 1993).

An excellent retention of amplificants was found with lengths ranging from 100 and 435 bp (CMV) to 640 bp (cy-tubulin) after reaction. Conversely, in the case of CMV, no significant ‘back-diffusion’ of very small (100 bp) amplificants into uninfected cells was noted using a commercially available biotinylated DNA probe. It is speculative as to

whether this is due to the size of the probe and/or biotin label, relative insensitivity of the biotin probe, or a charge effect. This is in contrast with ‘back-diffusion’ into cells by larger amplificants (196 bp) detected when using very small radiolabelled oligonu-


cleotide probes (Ray et al., 1991; Komminoth et al., 1992). It is suggested that large

(and charged) probes may penetrate membranes with difficulty, but after hybridization with a small target, may withstand washing steps with relative ease.

Despite the presence of natural inhibitors of PCR in body fluids (Innis and Gelfand,

1990; Khan et al., 1991), insufficient inhibitors remained after washing steps, to prevent

amplification; it is possible that preservation of cell membranes may prevent reaction

inhibitors from gaining access to target nucleic acid. a-Tubulin could only be detected by ISH after in situ PCR. In contrast, due to high

copy numbers in the case of CMV, we recorded a positive signal by ISH alone on

CMV-infected fibroblasts and CMV-infected urinary sediment, with a much stronger

signal after in situ PCR. Since sufficient amplificants were generated by our method,

cells were lysed without complicated extraction procedures to liberate PCR products,

which were used for independent confirmation of specificity. The results clearly demonstrate a close and predictable correlation with findings of ISH after in situ PCR.

The cells were thoroughly washed before lysis in order to prevent the possibility of

detecting extracellular formed amplificants.

Intracellular amplified gene sequences were successfully detected by ISH using single-stranded cRNA probes, a double-stranded DNA probe and sense oligonucleotide

probes. Oligonucleotide probes offer the important advantage that they can be generated to recognize sequences internal to the primers (Wolfe, 1988). Thus hybridization of

probes to the primers was unlikely for these oligoprobes, but cannot be absolutely excluded with other types of probes. Radiolabelled probes gave a more intense hy-

bridization signal, although they tended to obscure underlying morphology compared

with the biotinylated DNA probe. In situ PCR, followed by ISH, has many potential applications that may be of

particular value in cytological preparations. The detection of foreign (viral) DNA at the cellular level by this sensitive and specific method would permit the study and diagnosis

of, for example, either early or latent virus infection. This would be of use for evaluating transplantation specimens and in the study of virus-associated disease (e.g. Epstein-Barr

virus and lymphomas). The effect of treatment for CMV might also be monitored by this method in routine histological practice. Although further work is necessary, and other models need testing, we believe that in situ PCR may soon be of immense value in both

clinical practice and in research. However, vigorous controls must be used for evaluating results.

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