Development of Oligonucleotide Primers and Probes against ...

Vol. 30, No. 9

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1992, p. 2279-2283

0095-1137/92/092279-05$02.00/0 Copyright X 1992, American Society for Microbiology

Development of Oligonucleotide Primers and Probes against Structural and Regulatory Genes of Human Immunodeficiency Virus Type 1 (HIV-1) and Their Use for Amplification of HIV-1 Provirus by Using Polymerase Chain Reaction MAGDY R. DAWOOD,l 2* ROBERT ALLAN,' KEITH FOWKE,2 JOANNE EMBREE,2 AND GREGORY W. HAMMOND' 2 Cadham Provincial Laboratory, Winnipeg, Manitoba, Canada R3C 3Y1,' and Department of Medical

Microbiology, Faculty of Medicine, University ofManitoba, Winnipeg, Manitoba, Canada R3E oW32 Received 12 December 1991/Accepted 3 June 1992

The polymerase chain reaction is a powerful technique for amplifying a few copies of double-stranded genetic material to millions of copies in a few hours. The sensitivity and specificity of the polymerase chain reaction technique depend to some extent on the nucleotide sequences of the oligonucleotide primer pair used in the amplification. We report new oligonucleotide primers and probes which can be used for the amplification and detection of human immunodeficiency virus type 1 provirus sequences of not only structural but also regulatory genes. These primers are very sensitive and specific and can be used for the detection of African and North American strains of human immunodeficiency virus type 1.

for the amplification and detection of different strains of HIV-1. These primers and probes were found to be sensitive and specific for the amplification of African and North American strains of HIV-1.

Human immunodeficiency virus (HIV) is a retrovirus and is recognized as the cause of AIDS (11). HIV type 1 (HIV-1) is more prevalent than HIV-2 in North America, Europe, and Central Africa (24). Several methods have been developed for the detection of HIV infection. Virus isolation by cocultivation of peripheral blood lymphocytes (PBLs) with susceptible uninfected cells is time-consuming (9, 17, 21, 30). The detection of anti-HIV antibodies is a widely used technique for the diagnosis of infected individuals. However, this method does not differentiate between infection in newborns and passive carriage of maternal antibodies (26). The detection of viral RNA or the provirus by in situ hybridization or Southern blotting is not very effective (15, 16) because of the low number of copies of the viral RNA or provirus present in the specimen. An amplification step is required before testing. The development of a polymerase chain reaction (PCR) technique by Saiki et al. (27) for in vitro amplification of DNA has been successfully applied for the amplification of HIV provirus (1, 2, 7, 23, 28). PCR has subsequently been used to amplify the HIV genome (viral RNA) (3, 18). PCR products are analyzed by electrophoresis, and HIV-1-complementary sequences are detected by using 32P-radiolabeled probes or nonradioactive oligonucleotide probes (1, 23, 25). Design of specific oligonucleotide primer pairs for the amplification of a specific DNA sequence is considered an important step in reducing the nonspecific amplification of nontarget sequences. Crosscontamination or carryover between specimens can cause false-positive PCR results. False-negative PCR results can be obtained for several reasons, such as poor-quality DNA, low numbers of copies of the HIV provirus, and genetic variation in the region targeted for amplification (23). The last cause of false-negative PCR results can be overcome by using more than one primer pair (6). We report the sequences of five different oligonucleotide primer pairs and five corresponding oligonucleotide probes *

MATERIALS AND METHODS MgCl2 titration assay. The optimum concentration of MgCl2 in the PCR mixture was determined by using concentrations of MgCl2 that ranged from 0.1 to 2.0 mM. PCR was carried out as described below, and the optimum MgCl2 concentration was determined on the basis of the strongest signal obtained after dot blotting and hybridization with a 32P-radiolabeled probe. The optimum concentration of MgCl2 was determined to be 1.5 mM. Oligonucleotide primers and probes. Five pairs of oligonucleotide primers and five corresponding probes were designed (Table 1) to amplify HIV-1 provirus by the PCR technique. These primers and probes met six important criteria. First, they amplify regulatory and structural genes of HIV-1. Second, they are selected from conserved sequences among several strains of HIV-1 (22). Third, primers are 22- to 26-mers with G+C contents of 44 to 55% and melting temperatures (Tins) above 60°C. Relatively high Tins are used to ensure stable annealing between primers and the DNA template at a temperature close to the optimum required for Taq polymerase (72°C). Fourth, the 3' ends of the primer pair are not complementary to each other, to avoid the formation of a primer dimer (14). Fifth, each primer pair flanks a region in the range of 149 to 199 bp, a size that is considered good for amplification (20). Last, each probe has at least one restriction endonuclease site. Subjects and cell lines. Heparinized whole-blood specimens were collected from 10 HIV-1-seronegative individuals with no known risk factors for HIV-1 infection, 16 North American and 11 African HIV-1-seropositive individuals, and 4 North American HIV-1-seronegative individuals with risk factors for HIV-1 infection. The seronegative individu-

Corresponding author. 2279

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TABLE 1. Oligonucleotide primers and probes Primer or probe

Primers CPL el CPL e2 CPL gl CPL g2 CPL pl CPL p2 Nef a Nef c Vif a Vif c

Gene target

env env gag gag

pol pol nef nef vif

vif

Sequence

(5'-.3')

dGTTCCTTGGGTTCTTGGGAGCA dCTTGCTTGGAGCTGCTTGATGC dGACAAGGACCAAAGGAACCCTTTAGA dACTCCCTGACATGCTGTCATCATTTC dGTTCAATTAGGAATACCACATCCCGC dTTCCATCCCTGTGGAAGCACATTGTA dACCTCAGGTACCTTTAAGACCAATG dTGTGTAGTTCTGCCAATCAGGGAA dATTGTGTGGCAAGTAGACAGGATGA dCCTAGTGGGATGTGTACTTCTGAA

Probes CPL ep env dACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCACC CPL gp gag dAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACT CPL pp pol dGAAATCAGTAACAGTACTGGATGTGGGTGATGCATAT Nef b nef dGATCTCAGCCACTTTTTAAAAGAAAAGGGGGGACTG Vif b dAGTTTAGTAAAACACCATATGTATGTTTCA vif a The location is with reference to the HIV-1 HXB2 strain (22) sequence.

als with high-risk factors were one pediatric patient born to an HIV-seropositive mother, one health worker exposed to HIV-1-contaminated blood, and two adult males with other risk factors. PBLs were isolated from each blood specimen as indicated below in the section on specimen preparation. MOLT cells, a T-cell leukemia, CD4-positive cell line maintained in RPMI 1640 medium supplemented with 20% fetal calf serum, were used as negative control cells. The 8E5/ LAV (8E5) cell line, which has a single copy of HIV-1 provirus per cell and is defective in the production of infectious virus particles (10), was donated by the AIDS Research and Reference Reagent Program (ERC Bioservice Corporation, Rockville, Md.) and was used as a positive control. Specimen preparation. Blood specimens were collected in Vacutainer tubes containing heparin or EDTA. The tubes were left standing for 2 to 3 h, and the buffy coats (5 to 10 ml) were transferred to 15-ml centrifuge tubes and carefully underlaid with 3 ml of Ficoll-Hypaque. Specimens were centrifuged at 500 x g for 20 min by using a TJ-6R Beckman centrifuge. PBLs were collected at the interphase. PBLs were washed two times with phosphate-buffered saline (PBS) and counted by using a hemacytometer. PBLs (3 x 106 to 6 x 106 cells) were resuspended in 25 ,ul of doubledistilled H20 and 25 ,ul of 2% Triton X-100 in 20 mM Tris HC1 (pH 7.5)-2 mM EDTA. Cell lysates were incubated in boiling water for 15 min, cooled on ice, clarified by centrifugation, and stored at -20°C (4). Ten-microliter aliquots of cell lysates were used for amplification. Uninfected MOLT cell lysates were prepared as described above for the PBL cell lysates and were used as negative controls. Known numbers (5 to 100) of 8E5 cells were mixed with 2.5 x 105 PBLs isolated from seronegative individuals with no risk factors for HIV-1 infection. Cell lysates were prepared as described above. The 8E5 cell lysates were used for determining the sensitivity of the PCR developed in this study, and a preparation of 8E5 cells was used as a positive control in each PCR run. PCR. (i) DNA amplification. Ninety microliters of the reaction mixtures containing 10 ,ul of lOx PCR buffer (100 mM Tris-HCl [pH 8.0], 500 mM KCl, 15 mM MgCl2, 1% gelatin), 0.2 mM (each) deoxynucleoside triphosphates, 50

Location'

1554-1575 1715-1736 857-882 1033-1058 733-758 901-926 212-236 343-366 25-49 156-179

1609-1648 958-997 771-810 256-291 76-106

pM (each) primer, 2 U of Taq polymerase, and doubledistilled H20 was placed in 500-pl Eppendorf tubes with screw caps; and the mixtures were overlaid with 2 drops of mineral oil to prevent evaporation. Ten microliters of cell lysates was added to each PCR mixture, and the solution was incubated at 70°C until the start of the PCR cycles. PCR was carried out for 30 cycles; each cycle was 1 min of incubation each at 95, 58, and 72°C for denaturation, annealing, and elongation, respectively. At the end of 30 cycles, the reaction mixtures were incubated at 72°C for an additional 7 min for the completion of the extension of the PCR products. Each PCR run included a positive control specimen, which contained 10 copies of HIV-1 provirus, and two negative controls, a cell lysate from seronegative individuals with no risk factors for HIV-1 infection and a PCR mixture without any DNA template. (ii) Primer sensitivity assay. The sensitivity of each of the five primer pairs was determined by using the 8E5 cell line. Known numbers (5 to 100) of 8E5 cells were mixed with 2.5 x 105 PBLs isolated from seronegative individuals with no risk factors for HIV-1 infection. Cell lysates were prepared, and PCR was carried out as described above. The PCR products were analyzed for the presence of HIV-1 provirus sequences by Southern blotting and membrane hybridization with the corresponding 32P-labeled probe. (iii) Primer specificity assay. The specificity of each of the five primer sets was tested as follows. PBLs from 10 seronegative individuals with no risk factors for HIV-1 infection were separated, and cell lysates were prepared as described above. Each of the 10 specimens was amplified with all five primer pairs by using the PCR protocol described above. (iv) Analysis of PCR products. PCR products were analyzed by electrophoresis and then by Southern blotting and membrane hybridization. Ten-microliter aliquots of PCR products were subject to electrophoresis on 3% agarose and were transblotted by capillary transfer in 10x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) to a Zeta-Probe membrane (Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada). The membrane was briefly rinsed with 2x SSC. The DNA was immobilized on the membrane by baking at 80°C for 30 min. Prehybridization was carried out

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in 5 x SSC-20 mM NaH2PO4-7% sodium dodecyl sulfate (SDS)-10x Denhardt's solution-100 jig of denatured herring sperm DNA per ml at 50°C for 4 to 12 h. Hybridization was carried out at Tm - 10°C for 4 to 12 h in the same prehybridization solution, and 106 cpm of oligonucleotide probe per ml was labeled with [-y-32P]ATP by using T4 polynucleotide kinase (Boehringer Mannheim Canada Ltd., Laval, Quebec, Canada). The membrane was washed three times for 20 min each time at Tm - 20°C. The initial wash was in 2x SSC-1% SDS, the second was in lx SSC-1% SDS, and the last was in lx SSC. The radiolabeled signal was detected by autoradiography at -70°C with X-Omat film (Eastman Kodak Co., Rochester, N.Y.) between two Quanta III intensifying screens. The results were scored on the basis of the strength of the signal obtained on the autoradiogram in comparison with the signal from a positive control specimen (10 copies of HIV-1 provirus). Liquid hybridization of PCR products with 32P-radiolabeled oligonucleotide probes was carried as described by Ehrlich et al. (8). Thirty microliters of PCR product was mixed with 20 ,ul of liquid hybridization buffer. The final hybridization mixture contained 0.15 M NaCl, 2.5 mM EDTA, and 250,000 cpm of 32P-labeled probe. Specimens were incubated in a 100°C water bath for 5 min and then at 58°C for 30 min. Specimens were analyzed by 10% polyacrylamide gel electrophoresis, and the hybridized PCR products were visualized by autoradiography. A specimen was considered negative if no signal was detected. Positive specimens were divided into three groups on the basis of the visual density of the signal on the autoradiograph film. A specimen was considered + if the signal was less than that of the positive control, + if the signal was equal to that of the positive control, and + + if the signal was stronger than that of the positive control. RESULTS

Sensitivity and specificity of the primers and probes. Five of the HIV-1 provirus present in a total number of 2.5 x 105 cells could be detected by autoradiography after amplification by using each of the five primers developed in this study. The specificity was 100% for all seronegative individuals with no risk factors for HIV-1 infection. Clinical specimens. (i) North American specimens. The results presented in Table 2 show that one specimen was found to be PCR negative with the CPL el and CPL e2 primer pair but positive with the other four primer pairs. Although all five oligonucleotide primer pairs were able to amplify to detectable levels provirus from the other 15 specimens, the strength of the signal depended on the primer pair used, and therefore, it presumably depended on the viral strain present in the specimen. The use of more than one primer pair was important for avoiding false-negative recopies

sults. Comparison of the 32P signal obtained after membrane hybridization of PCR products from 13 North American specimens amplified with each of the primer pairs developed in this study is shown in Fig. 1. (ii) North American seronegative specimens. PBL specimens from four HIV-1-seronegative individuals with known risk factors for HIV-1 infection were amplified with all five primer pairs. All four specimens were found to be PCR negative with all five primer pairs. Consecutive blood samples collected from the pediatric patient and the health care

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TABLE 2. Detection of PCR products amplified from crude cell lysates or phenol-extracted DNA by using different primers No. of samples with the following result':

Primer set

Cell lysates from North American seropositive individuals (n = 16) CPL el-CPL e2 CPLgl-CPL g2 CPLpl-CPL p2 Nef a-Nef c Vif a-Vif c DNA extracted from seropositive individuals in Africa (n = 11) CPL el-CPL e2 CPLgl-CPL g2 CPL pl-CPL p2 Nef a-Nef c Vif a-Vif c

++

++

2 9 9 10 8

1

4 5 3 2 4

9 2 4 4 4

8 4 7 5 2

2 1 3

1 1 1 3 2

7

5 3

Total

positive'

15 16 16 16 16

3 7 4 6 9

a PCR products were detected by Southern blotting or liquid hybridization. -, no PCR signal detected; ±, signal weaker than positive control; +, signal equal to the positive control; + +, signal stronger than positive control. b -, +, and + + were considered positive.

worker during the past year were found to be consistently PCR negative. (iii) African seropositive specimens. DNA extracted from PBLs isolated from 11 HIV-1-seropositive individuals from Africa were amplified by PCR by using the five primer pairs reported in Table 1. Our results indicate that the CPL el-CPL e2 and CPL pl-CPL p2 primer pairs have sensitivities of 27% and 36%, respectively, with African specimens, while the same primers have sensitivities of 93.7 and 100%, respectively, with North American specimens. The Vif aVif c primer pair has a sensitivity of 81.8 and 100% for the African and North American specimens, respectively. The CPL gl-CPL g2 and Nef 'a-Nef c primer pairs were able to amplify to detectable levels of HIV-1 provirus in 63.6 and 54.5% of the African specimens, respectively. The last two primer pairs were the only ones that gave strong positive signals with the African specimens (Table 2). An autoradiograph of African specimens amplified with our primer pairs is presented in Fig. 2. The quality of the DNAs of the African specimens used in this study was ensured by PCR amplification by using the cellular HLA-DQa gene as a target.

DISCUSSION All the primers described in this report were 100% specific for the samples tested. The five oligonucleotide primer pairs were able to amplify to detectable levels approximately five copies of HIV-1 provirus in the total genomic DNA of 2.5 x 10' cells. CPL gl-CPL g2 and Vif a-Vif c primers are more sensitive than SK38/SK39 primers (23) in amplifications of African strains of HIV-1. A PCR-positive signal was detected in 100% of the North American HIV-1 seropositive specimens amplified with CPL gl-CPL g2, CPL pl-CPL p2, Nef a-Nef c, and Vif a-Vif c primer pairs, while the CPL el-CPL e2 primer pair had a sensitivity of 93.7% (15 of 16

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DAWOOD ET AL.

CPL e1,e2

4.

0 *

.

,

.*

v

0

1*@*

.4 ~~~~~4 *

0

C PL g1,g2

*

Cont. +

+

CPLp1,p2

STATUS

ANTIBODY

PRIMERS

_

+

+

-

_

+

+

v

4

-

+

*

nef a,c

0

vif a,c

m

@**@.

FIG. 1. Comparison of the 32P signals obtained on X-ray film after membrane hybridization of PCR products analyzed on 3% agarose followed by Southern transfer.

specimens) with the same specimens. False-negative PCR results have been reported previously (5, 13, 17). Results of the study described here show how multiple primers may overcome differences in reactivities. PCR-positive data have been reported when DNA in specimens from HIV-1-seronegative individuals at high risk of infection was amplified (15, 18, 19, 29) and when a p24-negative specimen from a 2-month-old infant was amplified (2). For the amplification of DNA from African HIV-1-seropositive individuals, the primers developed in the study described here had sensitivities of 27.3 to 81.8%, which varied with the oligonucleotide primer pair used. This suggests a genetic disparity between African and North American specimens and sequence variability among the African strains. This result is in agreement with that of Grankvist et al. (12). Our primers and probes can be used to detect North American strains of HIV-1 and may form an important means of differentiating between African and North American strains of HIV-1. The difference in the signal obtained from different specimens amplified with the same primer may be due to very few changes in the sequence of the amplified provirus in the area of the binding of the primer(s). In such a case, lowering of the annealing temperature may increase the density of the signal. The amplification protocol eliminated the nonspecific amplification of other closely related DNA sequences, which may be present in the specimens.

PRIMERS

AFRICAN SPECIMENS

Cont.

CPLel e2 CPL

glg2

UPL pli, p" nef vif acc

z

_m

FIG. 2. Comparison of the 32p signals obtained after liquid hybridization of DNA specimens from seven HIV-1-seropositive individuals from Africa amplified by PCR. The hybridization mixtures were separated on 10% polyacrylamide gels and were then exposed to X-ray film.

ACKNOWLEDGMENTS This work was funded in part by the National Health Research and Development Program (NHRDP) of Canada, grant number 6607-1490-AIDS, by an NHRDP National Health Fellowship to Keith Fowke, and in part by the Manitoba Department of Health. The 8E5/LAV cell line reagent was obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID; 8E5/LAV was prepared by Thomas Folks.

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