Molecular and Cellular Probes (1992) 6, 245-249

Comparison of spot-blot and microtitre plate methods for the detection of HIV-1 PCR products B . Conway, 1,2* L. J . Bechtel, 2 K. A. Adler, 3 R. T. D'Aquila,2 J. C. Kaplan 2 and M . S . Hirsch2 'Department of Microbiology & Immunology, University of Ottawa, Ottawa, Canada, 2Infectious Disease Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 'DuPont Medical Products, Boston, Massachusetts, USA . (Received 2 December 1991, Accepted 28 December 1991)

We have compared spot-blot methodology with a recently developed rapid microtitre plate assay for the specific detection of HIV-1 PCR products . We have studied blood specimens isolated from HIV-1 infected individuals (48 asymptomatic and 56 symptomatic patients) . Mononuclear cells were isolated, lysed and processed for PCR . Both PCR product detection methods were carried out in parallel on all amplified samples . HIV-1 sequences were detected by spot-blot or microtitre plate hybridization in samples taken from 42/48 asymptomatic and 53/56 symptomatic subjects . Concordant results between the two detection methods were observed for 90 samples, with 81 positive and nine negative assays . On repeat evaluation of the 14 discordant samples, nine showed concordant positive results, near the limit of detection of the assay . Serial dilutions of ACH-2 cells were amplified, and the PCR products were detected using the microtitre plate assay, yielding semi-quantitative results . The sensitivity of this simple, rapid assay compares with that of more laborious DNA detection systems . This may become a useful tool in HIV-1 research and in the clinical care of seropositive individuals . KEYWORDS :

PCR, HIV-1, microtitre plate, spot-blot .

INTRODUCTION The polymerase chain reaction (PCR) has been applied extensively in clinical and research settings as a tool for the detection of HIV-1 .' -3 We have described a method for the detection of HIV-1 DNA in crude cell lysates using non-radioactive oligonucleotide probes .' This use of crude cell lysates as PCR substrates represents an important simplification in the procedure . However, the processing of samples following amplification often remains complex . In our original protocol, this consisted of immobilization of amplified material on a nylon membrane, prehybridization, hybridization and six separate wash steps prior to colorimetric detection . In this study, we have compared this spot-blot methodology to a recently

developed rapid microtitre plate assay for the specific detection of HIV-1 PCR products .

MATERIALS AND METHODS We have studied blood specimens isolated from HIV-1 infected individuals attending the HIV Clinic at the Massachusetts General Hospital . These included 48 specimens taken from asymptomatic patients and 56 from symptomatic patients (45 with AIDS-related complex (ARC) and 11 with AIDS) . In preparing specimens for amplification, mononuclear cells were isolated by Ficoll-Hypaque centri-

*Author to whom correspondence should be addressed. 0890-8508/92/030245 + 05 $03 .00/0

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© 1992 Academic Press Limited

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fugation of heparinized blood . An aliquot of 6 x 106 cells was washed twice in phosphate-buffered saline (PBS), then lysed in 50 .tl of 1 % Triton-X-100 in 10 mm (pH 7), 1 mm EDTA . The lysate was then boiled for 15 min, to inactivate proteases . After brief centrifuga-

was carried out using the microtitre plate assay . Following PCR, the reaction mixture was denatured by heating to 94 µ C for 15 min, then placed on ice to

prevent renaturation . An aliquot of 10

©l

of each

amplification . The PCR reaction was carried out in a total volume

sample was then added to a streptavidin-coated microtitre plate well (DuPont NEN Research Products, Boston, USA), with two wells serving as substrate blanks. The wells were coated by adding 100 gl

of 50

streptavidin (10 Vg ml - ') in 0 . 1

tion, 10

©l

©l

of this lysate was subjected to DNA

using standard reagents (GeneAmpKit, Per-

kin-Elmer-Cetus, Norwalk, USA) . Primers designated

POL-1/2, 5 chosen to amplify a 323 base pair segment in the pol region of the HIV-1 genome, were added in a concentration of 0. 5 pm each . The amplification protocol consisted of 40 cycles with a 1 min denaturation step of 94µC, a 1 min annealing step at 45 µ C and a 1 min extension step at 72 µ C . Positive amplification controls, included in each assay, consisted of HIV-BH10 cloned into a pBR322 vector (10'-

103 copies). The plasmid DNA was diluted in HIV-1seronegative mononuclear cell lysate to approximate the total DNA content of other samples. Negative controls consisted of lysates of cells isolated from the blood of a seronegative volunteer, as well as an aliquot of PCR reaction mixture amplified without sample addition . Following amplification, 5 .tl of the reaction mix-

ture was denatured in 200 gl 0 . 2

N

NaOH, half of

which was then used for product detection . Up to 30 samples at a time were immobilized on an 8 x 15 cm Gene Screen nylon membrane (DuPont NEN Research Products, Boston, USA). The membrane was then placed in a resealable pouch containing 1 ml of hybridization buffer [5 x SSC, 0. 5% bovine serum albumin (BSA), 1% sodium dodecyl sulphate (SDS), 0 . 5% polyvinylpyrrolidone (PVP)1 and incubated at 55 µ C for 10 min . An alkaline phosphatase-conjugated oligonucleotide probe complementary to amplified sequences was then added at a final concentration of 2 . 5 mm and hybridization was carried out at 55 µ C for 15 min . After serial washes at 50 µ C in 1 x SSC/1% SDS, 1 x SSC/1 % Triton-X-100 and at room temperature in 1 x SSC (twice, 5 min each), the membrane was placed in a fresh pouch in the presence of nitroblue tetrazolium, a chromogenic substrate for alkaline phosphatase . A sample was positive if any blue colour development was detected following a 60 min incubation in the dark at 37µ C . An assay was considered valid if the reaction mixture control and seronegative cell lysate control were negative and the plasmid control containing 10' HIV-1 copies was positive . If these conditions were not fulfilled, test sample results were not interpreted and the PCR reaction was repeated . In parallel, detection of the same amplified product

M

sodium carbonate

buffer, pH 9 .6. Following overnight incubation at room temperature, the wells were washed three times with 1% Triton-X-100 in PBS and blotted dry .

Sample wells were overlaid with 100

©l

hybridization

buffer (6 x SSC, 1 % Triton-X-100, 0 . 1 % BSA), containing a biotinylated oligonucleotide capture probe (10 ©na) and an alkaline-phosphatase-linked oligonuc-

leotide reporter probe (5

LM),

both of which are

complementary to separate regions of the amplified product (see Fig. 1) . Following incubation in the dark at 37µ C for 30 min, the plate was washed six times with 1 % Triton-X-100 in PBS and blotted dry . A chromogenic substrate for alkaline phosphatase was then added to each well, consisting of 100 pl of a solution of 1 x diethanolamine with 0 . 5 mg ml - ' para-nitrophenol phosphate (Kirkegaard and Perry Labs, Gaithersberg, USA) . The plate was then incubated in the dark at 37 µ C for 30 min and readings were taken using a plate reader (SLT Lab Instruments, Hillsborough, USA) at 405 nm . A sample was considered positive if the optical density was greater than twice the mean optical density of the substrate blanks . Only valid assays, as defined above, were interpreted . In separate experiments designed to assess the quantitative nature of the microtitre plate detection system, ACH-2 cells serially diluted in seronegative cell lysates were amplified according to the previously described PCR protocol . As these cells con-

tain a single integrated copy of the HIV-1 genome, 6 results can be plotted in the form of a standard curve, relating optical density to proviral copy number .

RESULTS HIV-1 sequences were detected by spot-blot or microtitre plate hybridization in 42/48 samples taken from asymptomatic seropositive subjects and 53/56 samples taken from subjects with ARC or AIDS . Concordant results between the two detection methods were observed for 90 samples, with 81 positive and nine negative assays (Table 1) . The remaining 14 samples yielded discordant results, with nine positive by spot-blot and five positive by microtitre plate detection . In all these samples, a signal was detected

Microtitre plate PCR detection

Table 1 .

Target DNA

247

Comparative results of spot-blot and microtitre

plate product detection methods following DNA amplification of cell lysates isolated from HIV-1 seropositive individuals Microtitre plate method Capture probe Reporter probe

Spot-blot

+

81

9

Method

-

5

9

near the limit of detection of the assay (10 HIV-1 proviral copies) . On repeat evaluation, nine showed concordant positive results, with two remaining positive by spot-blot detection and three by microtitre plate detection alone . With respect to the interpretation of microtitre Streptavidin coated support

plate hybridization results, the mean optical density of the substrate blanks was 0015-0 . 020. Thus, depending on the individual assay, samples with an optical density above 0 . 030-0 . 044 would be considered positive . The optical density of seronegative cell lysates, included in each assay, was noted to be 0.016-0028 . The optical density of representative positive samples was 0.042-0 . 223, with a resullt of 0731 having been obtained on one occasion .

Streptavidin coated support

Serial dilutions of ACH-2 cells were amplified, and the PCR products were detected using the microtitre

Fig . 1 . Microtitre plate detection assay for PCR products, using streptavidin-coated wells, a biotinylated capture probe and an alkaline phosphatase-linked reporter probe .

plate assay. Two assays from separate amplification experiments are illustrated in Fig . 2 . Optical density of the PCR product is plotted as a function of ACH-2 cell number. Although inter-assay variability is observed between samples carrying the same number of HIV-1

0. 2

0 . 15

0

I

2

3

4

5

Log ACH-2 cell number Fig. 2 . Results of amplification of serial dilutions of ACH-2 cells (10'-10 4 cells) following two separate PCR reactions with products detected using the microtitre plate assay . (Experiment 1, - •-; positive cut-off 0 . 040, negative control 0 .028; Experiment 2, -+-; positive cut-off 0. 030, negative control 0 . 017) .

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B. Conway et al.

proviral copies, each amplification reaction yielded semi-quantitative results with a plateau effect being observed at higher proviral loads (> 100 ACH-2 cells) .

DISCUSSION

excess of 90% compared with that reported by other groups.'," We then evaluated the microtitre plate assay as a quantitative detection method . Our initial results indicate that the assay is at least semi-quantitative over a wide range of proviral copies . The well described attenuation in the exponential rate of pro-

The polymerase chain reaction (PCR) is a powerful tool for the detection of HIV-1 . We have recently described a rapid, sensitive method for the processing of clinical samples and the detection of HIV-1 proviral copies using non-radioactive probes .' Other groups have also favoured the use of crude lysates over purified DNA in sample preparation,', ' and have successfully utilized non-radioactive probes for product detection .'," Most reported assays make use of traditional hybridization methodologies . Although

duct accumulation that occurs during late PCR cycles, 15 especially with higher substrate concentrations, precludes true quantitation over the entire substrate range studied in our experiments . Nevertheless, using the plate reader, objective, numerical results are obtained which are easily amenable to precise statistical analysis . This is a distinct advantage compared to other methods relying on comparative intensities of bands on autoradiographs . Some interassay variability was observed . This is not surprising in the light of the exponential nature of PCR and the

these are reliable, they remain fairly complex and time-consuming . Some of these assays have been described as quantitative, 71 whereas others, including our original methodology, are qualitative in nature . Quantitative microtitre plate assays have been used to great advantage to provide more precise measurements of HIV-1 antigens and antibodies" A similar technique, using biotinylated primers and a horseradish peroxidase-linked oligonucleotide probe, has been described for the detection of HIV-1 PCR products ." In our application, microtitre plate wells are coated with streptavidin . The denatured PCR product is then added in the presence of a biotinylated capture probe and an alkaline phosphataselinked reporter probe, both of which are complementary to separate regions of the amplified product . The use of two probes clearly improves the specificity of the assay. Following a simple wash step, a substrate for alkaline phosphatase is added to the wells for a 30 min incubation . Final results are then obtained using a conventional plate reader . The entire detection assay requires approximately 90 min, as compared to 180 min or more for our previously described spot-blot method . We compared the two assays for the detection of HIV-1 proviral sequences in 104 mononuclear cell samples taken from HIV-1 seropositive individuals . On initial evaluation, 95 samples (91 . 3%) were positive by one or both assays, with 14 of these samples being positive by one assay alone . On repeat evaluation, nine samples yielded concordant positive results, with five remaining discordant . The 14 samples which initially produced different results with the spot-blot and microtitre plate assays appeared to carry proviral copy numbers at or near the level of detection of our tests. Thus our results are not entirely unexpected . Our overall detection rate in

marked effect on final yield of minor variations in the concentrations of reaction components ." Such minor variations can occur between serial amplification reactions prepared by the same individual, even using the same reaction mixture elements . Work is currently underway in our laboratory to improve the microtitre plate assay and develop a truly quantitative and more sensitive test . This work involves the evaluation of more sensitive primers and probes allowing reliable detection of one to five proviral copies and the use of a lower number of cycles of amplification to eliminate the plateau effect observed in our experiments, especially over a physiological range of proviral loads . In conclusion, we have shown that a semi-quantitative microtitre plate assay can be developed for the detection of HIV-1 PCR products . The sensitivity of this simple, rapid assay compares with that of more laborious detection systems . This may become a useful tool in HIV-1 research and in the clinical care of seropositive individuals .

ACKNOWLEDGEMENTS The authors wish to thank Mrs . N . Quirouette and Miss S . Grise for expert editorial assistance . This work was supported in part by the Medical Research Council of Canada and NIH grants CA12464 and CA35020 .

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