ELSEVIER Journal of Virological Methods 65 (1997) 45-54

Journal of Virological Methods

Development of an improved product enhanced reverse transcriptase assay

Audrey Chang *, Jeffrey M. Ostrove, Robert E. Bird

Microbiological Associates, 9900 Blackwell Road, Rockville, MD 20850, USA

Accepted 29 November 1996

Abstract

A PCR based reverse transcriptase (RT) assay was developed that has 104-fold higher sensitivity than conventional nucleotide incorporation assays and allows discrimination between false positive results generated by cellular polymerases and positives resulting from authentic RT activity. Recently, several reverse transcriptase (RT) assays have been developed where a reverse transcriptase reaction is performed on an RNA template/DNA primer combination. A specific region of the cDNA product is then amplified by the polymerase chain reaction to increase the sensitivity of cDNA detection. These reverse transcriptase assays are up to 106-fold more sensitive at detecting retroviruses than conventional methods. The drawback to these assays with increased sensitivity is the ,increased incidence of false po,sitive results generated by cellular polymerases that can reverse transcribe. The MS2 bacterio- phage RNA template and primers from one of the recently developed assays were used as the basis to develop the assay. A simple high resolution agarose gel was used as the endpoint for the assay without compromising sensitivity. In addition, the pH of the RT reaction was lowered to pH 5.5, the RT incubation was 1 h, and protease inhibitors were added to the RT reaction components. These modifications yield an assay that can discriminate between authentic RT activity and contaminating cellular polymerases. 0 1997 Elsevier Science B.V.

Keywords: Reverse transcriptase; Retrovirus; Assay; PCR

1. Introduction

Retroviruses comprise a large number of viruses known to infect many species, including humans, and have been associated with neoplasia,

* Corresponding author. Tel.: + 1 301 7381000; fax: + 1 301 7381036; e-mail: achang@microbio.com

immunodeficiencies, leukaemias and lymphomas (Coffin, 1990). A feature of the retrovirus life cycle is the integration of the newly reverse tran- scribed viral DNA into the host cell genomic DNA, forming the provirus. The presence of the provirus can activate or inactivate cellular genes, possibly with genetic consequences. In addition, retroviruses have high mutation and recombina-

0166-0934/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved.

PII SO 166-0934(96)02168-4

46 A. Chang et al. 1 Journal of Virological Methods 65 (1997) 45-54

tion rates which potentially can alter host range and virulence. Safety testing for retroviruses of cell substrates used to produce biologicals is an important issue. The most frequent viral contami- nant found in these cell substrates are retroviruses (Moore, 1992; Losikoff et al., 1992). Therefore, it is of great importance to insure that biological products and vaccines are free of contaminating viruses.

Currently, the biotechnology industry uses three assays for the detection of retroviruses from cell substrates or biological products. These are transmission electron microscopy, growth of virus on sensitive cells and detection of reverse tran- scriptase activity. The detection of reverse tran- scriptase is an important part of this panel of assays since all retroviruses contain reverse tran- scriptase within the virion. A sensitive assay for RT is capable of detecting a virus which may not grow on the indicator cells being tested in virus propagation assays.

Numerous RT assays exist that measure the incorporation of radiolabelled deoxyribonu- cleoside triphosphates (e.g. 3H-TTP) into DNA using RNA as a template (Roy-Burman et al., 1976). These assays have been used for measuring RT activity in many laboratories. The incorpo- rated radiolabel is then measured by a scintilla- tion counter or by autoradiography. Recently, several polymerase chain reaction (PCR) based RT detection assays have been developed (Silver et al., 1993; Busso and Resnick, 1994; Pyra et al., 1994; Heneine et al., 1995) that are several orders of magnitude more sensitive than the incorpora- tion-based assays. These assays are similar funda- mentally, in that they detect RT activity by amplifying the newly synthesized cDNA from an RNA template using PCR. It has been shown that these RT-PCR detection assays can detect RT from various viral sources and from cell superna- tants with increased sensitivity of detection by 105-106-fold over conventional assays. However, coincidental with this increase in sensitivity is an increase in false positive reactions coming from contaminating DNA polymerases, such as poly- merase CI and y, which are known to have RT-like activity (Kornberg and Baker, 1992a,b).

We developed a PCR-based RT assay using the MS2 RNA template and primers from the assay developed by Pyra et al. (1994). Improvements were made to the method which significantly de- crease the level of false positives. It is shown that reduction in the pH of the RT reaction reduces false positives caused by cellular polymerases and yields an assay which is more specific and 104-fold more sensitive than conventional (non-PCR- based) RT assays.

2. Materials and methods

2.1. Reagents

Molecular biology grade chemicals were pur- chased from Sigma. Stock solutions were made in diethylpyrocarbonate (DepC) treated H,O. Protease inhibitors were purchased from Boehringer Mannheim and leupeptin and aprot- inin were resuspended in DepC treated H,O and Pepstatin was resuspended in methanol. A syn- thetic oligonucleotide that anneals to the MS2 RNA (GenBank accession no. 502467) from nu- cleotide 109-132 RT-1 [5’-d(CATAGGTCAAAC- CTCCTAGGAATG)-3’1 was used as a primer for cDNA synthesis. RT-2 [S-d(TCCTGCT- CAACTTCCTGTCGAG)-3’1 (Pyra et al., 1994) is the oligonucleotide that will anneal to MS2 nucle- otide sequences 21-33 used in the PCR reaction. Both oligonucleotides were purchased from Gibco/BRL.

2.2. Cell cultures

All cell lines were maintained in either DMEM or EMEM with serum (fetal bovine or newborn calf) with or without gentamycin.

2.3. Sources of RT

Squirrel monkey retrovirus (SMRV), Equine infectious anemia virus (EIAV), Rauscher mouse leukemia virus (R-MLV), Moloney mouse leukemia virus (MMLV), Gibbon ape leukemia virus (GaLV) and Adenovirus virus were all pre- pared at Microbiological Associates. Raji cells

A. Chang et al. /Journal of Virological Methods 65 (1997) 45-54 4-I

infected persistently with Simian Retrovirus-2 (SRV-2) H9 cells infected with Simian Im- munodeficiency Virus (SIV), and LLCMK2 cells infected with Foamyvirus were maintained at Mi- crobiological Associates as sources of reverse transcriptase. The Human T-cell Leukemia Virus- 1 (HTLV-1) producing MT-2 cells were the source for HTLV virus. A GaLV producing gibbon ape T cell lymphoma wa.s used as a GaLV source. A purified preparation of Rauscher murine leukemia virus (R-MLV) had an average titer of 4.3 x lo6 PFU/ml as determined by the XC plaque assay using SC-l cells (ATCC CRL-1404) and XC cells (ATCC CCL-165) as previously described (Kle- ment et al., 1969). The same preparation of R- MLV had a viral particle count of 2.56 x lo9 viral particles/ml as determined by electron microscopy (Advanced Biotechnologies, Columbia MD). Purified RTs were purchased commercially as fol- lows: Avian myeloblastosis virus (AMV) (Amer- sham), HIV (Worthington and Amersham), MMLV (Gibco/BR.L), Superscript II (Gibco/ BRL).

2.4. Sample preparation

2.4.1. Cell lysates Adherent cell lines were rinsed with ice cold

PBS (Dulbecco’s F’hosphate Buffer Saline (D- PBS), KC1 200 mg,/l, KH,PO, 200 mg/l, NaCl 8000 mg/l, Na3HP0,.7H,O 2160 mg/l) and scraped in 5 ml of ice cold PBS. Cells were centrifuged at 1OOCl x g and the resulting pellet was resuspended in 500 ,ul PBS (for T75 flask-ap- proximately lo6 cells) or 200 ~1 PBS (for T25 flask). Suspension cells were centrifuged at 1000 x g and washed once with ice cold PBS, centrifuged again and the resulting pellet was resuspended in either 500 or 200 ~1 depending on the flask size. Resuspended cell pellet (100 ~1) was added to 100 ~1 of 13uffer A (50 mM KCl, 25 mM Tris-HCl pH 7.5, 5mM DTT, 0.25 mM EDTA pH 8.0, 0.025% Triton X-100, 50% glycerol) + I(protease inhibitor cocktail containing final con- centrations of aprotinin 1 pg/ml, leupeptin 1 ,ugg/ml, pepstatin 0.‘7 pg/ml). Then, 4 ~1 of a 2% Triton X-100 solution was added to the cell mix- ture (0.04% final Triton X-100 concentration).

The samples were then frozen and thawed by changing the temperature from - 70 to 37°C. Total protein concentration was determined using the BioRad Protein assay kit (Bradford, 1976). The amount of cell lysate protein used varied between 0.2-5 ,ug. When two lysates are com- pared, the same quantity of lysate protein was used.

2.4.2. Cell supernatant Conditioned media from actively growing cells

were harvested to test for RT activity in cell supernatants. The media were on the cells for a minimum of 72 h before harvesting. Medium (1 ml) was centrifuged at 3000 rpm for 5 min to clear cellular debris. Then, 100 ~1 Buffer A + I was added to 100 ~1 of the cleared media (cell super- natant) and 4 ~1 of 2% Triton X-100 was added. The sample was frozen and thawed in the same manner as described above for the cell lysates.

2.4.3. Virus Purified virus was diluted in Buffer A + I with

0.04% Triton X-100 and the sample was frozen and thawed as described above. Following this, lo-fold dilutions were made in Buffer A + I with 0.04% Triton X-100.

2.4.4. Purl$ed RT For all the commercially available RTs used in

this study, 1 unit (U) is defined as the amount of enzyme required to incorporate 1.0 nmol of de- oxyribonucleotide into acid precipitable material in 10 min at 37°C using poly(rA)-oligo(dT),,_,,. The enzyme was serially diluted in Buffer A just prior to the assay.

2.5. PCR based RT assay

2.5. I. Reverse transcription Bacteriophage MS2 genomic RNA (Boehringer

Mannheim) was annealed with RT-1 essentially as described by Pyra et al., 1994 and used as the primer/template for the RT reaction. Primer/tem- plate was prepared by mixing, per reaction, 0.28 pmol (0.3 pg) of MS2 RNA with 10 pmol (72 ng) of primer RT-1 in H,O, heating at 85°C for 5 min and then immediately placing the tube at 37°C for

48 A. Chang et al. 1 Journal of’ Vimlogical Methods 65 (1997) 45-54

30 min. The annealed RNA/primer was then placed at 4°C for 5 min. Two separate RT buffers were freshly prepared that were identical except for the pH of the 56 mM Hepes (pH 4.5-8.3 or 56 mM Tris pH 8.3), 56 mM KCl, 9.2 mM MgC&, 11.2 mM DTT, 0.1 U/p1 re- combinant Rnasin (Promega), 0.13 pg/pl Bovine Serum Albumin (BSA)(New England Biolabs), 1 mM of each dNTPs (Promega). Primer/template is added to 23.6 ~1 RT buffer in a PCR tube. The RT source (ranging from 1 to 5 ~1) is added last. The mixture was incubated at 37°C for 1 h.

2.5.2. PCR ampl$cation To each RT reaction, 75 ~1 PCR mix [lo ~1

10 x PCR Buffer (Perkin Elmer), 1 ~1 dNTP mix (25 mM), 5 ~1 MgCl, (25 mM), 2 ~1 of RT-1 (10 pmol/hl), 3~1 of RT-2 (10 pmol/pl), 0.1 ~1 Rnase A (1 pg/pl), 53.9 ~1 H,O] with 7.5 U Taq polymerase (Perkin-Elmer) were added. The amplification conditions for the PE 2400 are as follows: 94”C/15 s of denaturation, 55”C/ 50 s for annealing, 72’C/55 s for extension for a total of 25 cycles and a final extension of 72”C/ 10 min.

2.5.3. Detection of PCR product The PCR amplification of the cDNA pro-

duces a 112 bp PCR product at the 5’ end of the MS2 RNA from nucleotide 21 to 132. The PCR product is directly visualized on a horizon- tal 2.5% Metaphor agarose gel (FMC BioProd- ucts). The agarose is dissolved in 1 x Tris-borate EDTA (TBE) (0.1 M Tris borate, 0.001 M EDTA) and ethidium bromide (0.4 pg/ml). The gel is chilled at 4°C for at least 30 min before use. Then 3 ~1 6 x gel loading dye (Novex) is added to 10 ~1 of the PCR product and the mix was loaded on the gel. An appropriate DNA size marker [123-bp ladder or lOO-bp lad- der (Gibco/BRL)] is also utilized. The gel is run at 100-150 volts for approximately 1 h. The DNA bands were visualized by placing the gel on a UV transilluminator and pho- tographed.

3. Results

3.1. Titration of purl$ed RT

A PCR based RT assay was developed using the MS2 RNA template and primers described by Pyra et al., 1994. Our assay includes the addition of protease inhibitors, reducing the RT reaction incubation time to 1 h, combining the separate RNase A digestion step with the PCR, and run- ning the PCR product on a high resolution agarose gel to visualize the amplification product directly (Fig. 1A). This RT assay was examined for sensitivity by diluting the recombinant M- MLV reverse transcriptase, Superscript II (Gibco- BRL), in lo-fold serial dilutions, and performing a 1 h RT reaction at pH 8.3. As shown in Fig. IB, the 112-bp band is produced in samples which contain RT. The detection limit for this assay, as defined by visualization of the 112-bp PCR band, can be see at 10 ~ 9 U of RT. The specific activity of Gibco-BRL Superscript II is 330000 U/mg (Lot No. EPF401). The molecular weight of Su- perscript II is 78 000 Da. Therefore, 10 - 9 U corresponds to 22 molecules of RT. Other purified RT enzymes, HIV RT, AMV RT, M-MLV RT, all had similar limits of detection of 10 - 9 and 10-r” U (data not shown). The negative controls of no RT and no MS2 RNA template did not have the 112-bp band, indicating that no contam- inating DNA or contaminating RT activity from Taq or Rnasin was present. The first control, no RT, was important to demonstrate that the RNase step could be included with the PCR step since Taq polymerase is known to have low re- verse transcriptase activity. The sensitivity ob- tained with the high resolution agarose gel is comparable to the ELISA and Southern blot methodology (Pyra et al., 1994) and is preferred for its simplicity.

3.2. Retrovirus infected and non-infected cell culture supernatant and cell lysate

Cellular DNA polymerases that reverse tran- scribe, such as CI and y, can, under certain condi- tions, utilize an RNA template and generate false positive results in these assays. Therefore, PCR

A. Chang et al. /Journal of Virological Methods 65 (1997) 45-54 49

m MS2 phag.? RNA

+ RT-1

+TEST ARTICLE

I

Tris pH 8.3

enzyme 37 qc 5 hours

MQ2+ virus KCI cell supernatant dNTPs cell 1ysate RNAsin

B MS2 phaQe RNA

1 RT-1

1

RNaseA

1 Taq polymerase RT-2 primer

RT-2 +

112 bp PCR product

ENZYME

‘112 bp ---)

primers ----+

Fig. 1. (A) Schematic diagram of the RT-PCR Assay: (Pyra et al., 1994). The thin wavy line represents the MS2 phage RNA template which the RT-1 primer (short bold arrows) anneals. The RT components are listed and the newly synthesized cDNA is depicted by the horizontal bold line. The PCR components are then added and the conditions for PCR listed. (B) Titration of purified RT with the RT-PCR assay A recombinant M-MLV RT (Superscript II, Gibco-BRL) was diluted and tested. The numbers above each lane refer to the total units of RT tested. The samples were electrophoresed on a 2.5% Metaphor (FMC) agarose gel and the 112-bp PCR product amplified from the cDNA is indicated by the upper arrow. Primers are shown with the lower arrow.

based RT detection assays have been limited to can infect and reverse transcribe its genome. The detecting retrovirus in ‘clean’ culture supernatant data in Fig. 2 demonstrate that both the cell which contain little or no cellular polymerases. supernatant (DAP lane 1) and cell lysate (DAP This point is illustrated in Fig. 2. The 72 DAP lane 2, prepared as described above), when tested (DAP) cell line is ‘derived from an NIH 3T3 cell for RT activity in the presence of Mg+ + at pH line and produces recombinant retroviruses that 8.3, are positive. This is expected since the retro-

50 A. Chang et al. 1 Journal of Virological Methods 65 (1997) 45-54

~2 DAP NIH3T3 -7 1234512345M

112bp ---_)

Fig. 2. Cell lysate and cell supernatant of uninfected and retrovirus-infected cell lines. Lane (1) cell lysate, (2) cell super- natant, (3) cell supernatant with 1O-4 unit RT (Superscript II), (4) cell lysate without RNA, and (5) cell supernatant without RNA template.

virus is present in the DAP cell supernatant and the cell lysate. The uninfected NIH 3T3 cell super- natant was negative for RT in the assay (NIH lane 2) whereas the NIH 3T3 cell lysate (NIH lane 1) was positive. Lanes 4 and 5 are cell lysate and cell supernatant without RNA template, respec- tively. Since the NIH 3T3 cell line does not har- bor infectious retrovirus and expression of full length endogenous virus IAPs is low (Grigoryan et al., 1985), the ‘RT’ activity seen in the NIH 3T3 cells was assumed to be a result of cellular DNA polymerases activity. Several other unin- fected cell lysates also tested positive under the same assay conditions (Table 1).

Table I Testing various uninfected cell lysates for RT activity

Cell line pH 8.3 pH 5.5 pH 5.5+spike

293 + NIH 3T3 + Raji + MRC-5 + HeLa + CHO + BHK + LLC-MK2 += H9 +” Mm dunni fa

- ND - + - + - + - + - + - + - + - + - +

The spike used was lop3 or 1O-4 U of AMV RT “Weak Positive.

3.3. The effect of pH in the RT reaction mix

It became clear even though steps can be taken to minimize cellular polymerase contamination in cell preparations (low speed centrifugations, and harvesting cell supernatants from healthy cul- tures) it is not possible to totally eliminate cellular polymerases that may be released from lysed cells. We investigated conditions of temperature and pH that might allow discrimination between viral RT and cellular polymerase activity. The perfor- mance of the RT reaction at reduced pH yielded results. Cell lysates from the uninfected NIH 3T3 cells and y2 DAP cells were tested for RT activity at varying pHs from 3.5 to 6.8 as described. The results are shown in Fig. 3A. The RT activity in the uninfected NIH 3T3 lysates disappears below pH 6.5 whereas the activity in the DAP lysate where retroviruses are present does not disappear until the pH is lower than 4.5. Cell supernatants from both cell lines were subjected to a pH titra- tion analysis pH 4.5 to 8.3 (Fig. 3B). There is no RT activity in NIH 3T3 cell supernatant at any pH. However, there is activity in the ‘~2 DAP supernatant at all pH’s tested resulting from the virus produced by these cells.

3.4. Uninfected cell lysates and retrovirus

Along with the NIH 3T3-y2 DAP cell line pair, cell lysates from two additional uninfected/in- fected cell line pairs (Raji and SRV infected Raji, H9 and SIV infected H9) were tested for RT in the same manner. Only the cell lines that harbor infectious retrovirus produced the 112-bp band with the low pH RT reaction whereas the unin- fected cell lines did not (data not shown). Table 1 summarizes the results of additional uninfected cell lysates from various sources that were pre- pared and tested. Although a false positive for reverse transcriptase was seen at the higher pH RT incubation, all cell lines tested negative for RT activity at pH 5.5. The negative results are not an outcome of inhibition of RT since the presence of an RT spike results in a positive signal.

In addition to SIV and SRV, Table 2 lists the other retroviral RTs we have detected in our assay. Foamyvirus, human T-Cell leukemia virus

A. Chang et al. 1 Journal of Virological Methods 65 (1997) 45-54

PH

112bp +

112bp --+

Nlt

2 DAP

51

T3

112 bp+

112 bp+

NIH3T3

y2 DAP

Fig. 3. (A) Effect of decreasing pH in the RT reaction mix (NIH3T3 and y2 DAP) cell lysate and (B) cell supernatant. Cell lysates and cell supernatant from the uninfected NIH 3T3 cells and the y2 DAP cells were tested for RT at varying pHs. The sample without RNA serves as the negative control (neg) and the lOO-bp marker (M) are indicated.

52 A. Chwg et al. / Journal of Virological Methods 65 (1997) 45-54

Table 2

Testing various retrovirus for RT activity

Virus or RT spe-

ties

Source Results

Foamyvirus

HTLV I

GaLV

SIV

SRV 2

SMRV

EIAV

R-MLV

MMLV

AMV

HIV

MMLV

Adenovirus

Infected cells +

Infected cells +

Infected cells + Infected cell supernatent +

Infected cell supernatent +

Purified virus +

Purified virus + Purified virus + Purified virus + Purified RT + Purified RT + Purified RT +

Purified virus y _

type I, squirrel monkey retrovirus, equine infec- tious anemia virus, avian myeloblastosis virus, human immunodeficiency virus, Gibbon ape leukemia virus, representing the different retro- virus subfamilies (Oncovirus, Lentivirus, and Spumavirus), all tested positive for RT activity in the low pH RT-PCR assay. Adenovirus viral par- ticles, a double stranded DNA virus, which does not contain RT, tested negative for RT activity in the assay as expected.

3.5. Sensitivity of detection of Rauscher MLV

using the PCR based RT assay

A purified preparation of R-MLV viral parti- cles with an average titer (determined by the XC plaque assay) of 4.3 x lo6 PFU/ml and a viral particle count by electron microscopy of 2.56 x lo9 particles/ml was serially diluted and 5 ~1 was tested for RT at pH of 8.3 (Fig. 4A) and pH 5.5 (Fig. 4B). At the higher pH, the limit of detection was at 10 - 6 which corresponds to 0.021 infectious particles (10.3 viral particles). At the lower pH, the limit of detection was at lob4 which corre- sponds to 2.1 infectious particles.

3.6. Titration of purified RT using the PCR based RT assay

A 2 log difference in assay sensitivity was noted between the two pH’s. This was also observed for

all RTs tested, including AMV, M-MLV, and HIV (data not shown). With the assay performed at the higher pH, the RT activity of these enzymes titrate to 10 - 9 U (approximately 4 x lo2 RT molecules for HIV RT) (data not shown). Fig. 5 shows HIV RTs from two commercial sources that were serially diluted and tested at pH 5.5 in the presence of 0.5 pg of BSA. Each HIV RT had similar limits of detection of lo-’ U (approxi- mately 4 x lo4 molecules RT). Nevertheless, the low pH RT-PCR assay is significantly more sensi- tive than the radiolabel-based RT incorporation assays.

3.7. Effect of decreasing pH in the RT reaction

mix comparing cell lysate and cell lysate with

retrovirus spike

Cell lysate was prepared from NIH 3T3 cells

Dilution of Virus

A.

112 bp+

B.

112 bp-k

Fig. 4. Titration of RT activity from purified Rauscher MLV

by changing the pH of the RT reaction. RT activity with a

reaction performed at (A) pH 8.3 or (B) pH 5.5. A sample

without RNA (neg) serves as the negative control and (M)

corresponds to the 123-bp ladder.

A. Chang et al. /Journal of Virological Methods 65 (1997) 45-54 53

units

112tIp-w

112bp-k

Fig. 5. Titration of HIV RT using the pH 5.5 RT reaction

HIV RTs from two commercial sources were serially diluted

and tested at pH 5.5 in the presence of 0.5 pg of BSA. The

123-bp marker (M) and the no RNA negative control (neg) are

indicated.

and total protein content was determined. Then, 200 ng of total protein from the NIH 3T3 cell lysate was tested in RT reactions at different pHs ranging from pH 8.3 to 4.5 (Fig. 6, lanes 1). As the pH of the RT reaction is reduced, the intensity of the 112-bp band decreases to the point where at pH 5.5 and below, a band is no longer evident. In contrast, when the same cell lysate now con- taining an R-MLV virus spike (Fig. 6. lanes 2) is tested over the salme range of pH, the 112-bp band is evident at all the pH’s tested, including pH 5.5 and below. ‘The R-MLV virus tested alone over the range of pHs produces the same pattern (Fig. 6, lanes 3). The reverse transciptase activity displayed by authentic RT and cellular poly- merases both shov+ a pH optimum at the higher pHs of 8.3 and 7.5, respectively. However, it is evident that the plH activity range for authentic reverse transcriptase activity is broader than that for cellular polymerase reverse transciptase activ- ity. At the lower pH range, a signal is clearly produced only in samples where reverse transcrip- tase is present.

4. Discussion

There are only two retroviral subfamilies that are known to be pathogenic for humans (Cann and Chen, 1990; Hirsch and Curran, 1990). These are the leukemia viruses, HTLV-I and HTLV-II and the immunodeficiency viruses, HIV-l and HIV-2. However, it is clear that there are many retroviruses that possess a wide host range and may pose a threat to the human population. The viruses may be either primate or non-primate in origin such as the murine amphotropic and xenotropic retroviruses (Coffin, 1990). Therefore it is prudent to test for retroviruses that may be present in biological products made in mam- malian cells. These include both primary cell cul- tures and continuous cell lines. Such tests could also be used as diagnostics for retrovirus infection by the presence of RT in human blood or other fluids and tissue biopsies.

The successful analysis of the retroviral status in biological samples depends upon the interpreta- tion of data generated from several assays (co-cul- tivation, EM, RT). The RT-PCR assays exploit the extreme sensitivity of PCR to detect retro- viruses by the presence of reverse transcriptase activity. However, the low level of RNA depen- dent DNA polymerase activity from cellular poly- merases can mask authentic RT activity and produce invalid results in the RT-PCR assay. In this study, we have developed modifications of the RT-PCR assay that will detect authentic RT ac- tivity at a sensitivity level several logs greater than that obtained with the conventional assays while eliminating the false positives generated by cellu- lar polymerases. The most significant modification was to lower the pH of the RT reaction and when the low pH RT-PCR assay was challenged with crude lysate from uninfected growing cells that contain all forms of cellular polymerases, discrim- ination between authentic RT activity from cellu- lar polymerases was possible. The ease of preparing samples (one template, one divalent cation, no radioactive/organic reagents, no ultra- centrifugation) and the wide range of retrovirus RT that can be assayed gives the low pH RT- PCR assay considerable potential for use as a screening procedure for retroviruses.

54

PH

A. Chang el al. / Journal qf’ Virological Methods 65 (1997) 45-54

112 bP --,

8.3 7.5 6.5 5.5 4.5 nnnn I

12341234123412341234M

1 = cell lysate (NIH 3T3) 2 = cell lysate + R-MuLV (40 pfu) 3 = R-MuLV (40 pfu) 4 = negative control (no RNA template)

Fig. 6. Effect of decreasing pH on reaction inhibition rates comparing cell lysates with cell lysates spiked with virus. Gel electrophoresis analysis: (1) cell lysate, (2) cell lysate spiked with R-MLV (2560 viral particles or 40 PFU), (3) R-MLV virus (2560 viral particles), (4) cell lysate with no added RNA template (negative control).

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