AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 16, Number 15, 2000, pp. 1503–1505 Mary Ann Liebert, Inc.
Short Communication Development of a One-Tube Multiplex Reverse Transcriptase-Polymerase Chain Reaction Assay for the Simultaneous Amplification of HIV Type 1 Group M gag and env Heteroduplex Mobility Assay Fragments FATIM CHAM,1,2 LEO HEYNDRICKX, 1 WOUTER JANSSENS, 1,3 KATLEEN VEREECKEN,1 KATHLEEN DE HOUWER, 1 SANDRA COPPENS,1 GERT VAN DER AUWERA,1 HILTON WHITTLE, 2 and GUIDO VAN DER GROEN1
ABSTRACT The emergence of intersubtype recombinant HIV-1 isolates has made it imperative to analyze different regions of HIV-1 genomes. For this purpose a one-tube multiplex RT-PCR, coamplifying first-round amplicons that allow amplification of gag and env heteroduplex mobility assay (HMA) fragments from different HIV-1 group M isolates, was developed, starting with plasma samples. The multiplex RT-PCR assay is sensitive: 115 of 136 (84.5%) samples were positive for both gag and env, positive amplification of the gag fragment was observed in 130 of 136 (95.6%) samples, while for the env fragment 119 of 136 (87.5%) tested positive. The multiplex RT-PCR in combination with gag and env HMA makes large-scale HIV-1 subtyping fast, simple, and more economical.
T
of a reliable and economical screening technique to track prevalent HIV-1 subtypes and emerging recombinant isolates worldwide is important in the effort toward developing a protective vaccine. Although full-length sequencing remains the most accurate technique to characterize viral genomes, it is technically challenging, and expensive, for use in large-scale studies, especially for developing nations that are mostly affected by the HIV epidemic. The heteroduplex mobility assay (HMA) based on analysis of selected regions in the env gene (env HMA) allowed for rapid subtyping of HIV-1 under field conditions.1 An env HMA kit was developed and widely distributed through the NIH and the WHO/UNAIDS. A series of training workshops, organized by the WHO/UNAIDS, was held in developed and developing countries to ensure an appropriate use of this technique. Various laboratories have successfully used the env HMA, generating important information on the molecular epidemiology of HIV-1. The scientific eviHE DEVELOPMENT
dence of an increasing global spread of recombinants2 dictated a need to extend the env HMA method to other parts of the HIV-1 genome, increasing probabilities of detecting HIV-1 recombinants. For this purpose a gag HMA was developed.3 In addition to the env HMA kit, the NIH AIDS Reagent Program (Rockville, MD) foresees integration of a gag HMA kit. Hence the gag/env heteroduplex mobility assay (gag/env HMA)1,3,4 remains the method of choice for rapid screening under field conditions of prevalent subtypes, circulating recombinant forms (CRFs),5 and intersubtype recombinant forms (IRFs)5 in developing countries where vaccine trials are being prepared. We developed a cost-effective and less labor-intense multiplex RT-PCR assay that can simultaneously generate firstround amplicons that allow amplification of gag and env HMA fragments from different HIV-1 group M plasma samples. An evaluation panel was available, consisting of 136 plasma samples of HIV-1 group M (A–H), CRF01.AE-, and CRF02.
1 Department
of Microbiology, Institute of Tropical Medicine, B-2000 Antwerp, Belgium. Research Council Laboratories, Banjul, The Gambia. 3 Flanders Interuniversity Institute for Biotechnology, VIB, B-9052 Ghent, Belgium. 2 Medical
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CHAM ET AL. TABLE 1.
HIV-1 Group M
CRF IRF
MULTIPLEX RT-PCR ASSAY
Subtypea (gag/env) A/A#b B/B C/C D/D F/F G/G H/H CRF01.AE/A# CRF02.AG/A #
AND
N 23 30 16 11 3 14 4 2 21 12 Total: 136
AMPLIFICATION
OF
gag/env HMA FRAGMENTS
gag
env
gag and env
22 30 16 10 1 14 3 2 20 12 130 (95.6%)
21 28 12 9 1 14 3 0 20 11 119 (87.5%)
20 28 12 8 1 14 2 0 19 11 115 (84.5%)
Abbreviations: N, Number of plasma samples; CRF, circulating recombinant form; IRF, intersubtype recombinant form. a Subtypes for env were based on the 700-bp (ES7/ES8) HMA fragments encoding V3–V5,1 and subtypes for gag were based on the 460-bp (H1G1584-G17) HMA fragment encoding amino acids 264–417 (according to HIV-1 HXB2_Pr55; http://hiv-web.lanl.gov/).3 b A# , Subtype A could not be differentiated from CRF02.AG by env HMA. AG-infected patients from different geographic regions (Bénin, Cameroon, Kenya, Zambia, Belgium), for which a monoplex reverse transcriptase-polymerase chain reaction (mono-RTPCR) resulted in 100% sensitivity for both gag and env fragments, and for which the gag/env subtype was determined by HMA (Table 1). Viral loads were unknown for samples taken in African countries and ranged from 5 3 103 to .7.5 3 105 for samples taken from Belgian individuals. Total RNA was extracted6 starting from 100 ml of plasma. The RNA was stored in 50 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8) at 270°C. One-tube multiplex RT-PCR was performed in a 25-ml reaction volume. To avoid formation of nonspecific PCR fragments, two separate mixtures, stored on ice, were prepared. Mix 1 (15-ml volume) was composed of nuclease-free water, 1.0 mM MgSO4, dATP, dCTP, dGTP, and dTTP (0.2 mM each) (Access RT-PCR; Promega, Leiden, The Netherlands), gag primers H1G777 and H 1P202,3 and env primers ED5 and ED12,1 at a concentration of 1.0 mM each, and 3 ml of template RNA. Mix 2 (10-ml volume) contained 2.5 units of avian myeloblastosis virus (AMV) RT, 5 units of Tfl DNA polymerase, 53 AMV/Tfl reaction buffer, and nuclease-free water (Access RT-PCR; Promega). After pooling both mixtures, RTPCR was done at 48°C for 45 min, followed by inactivation of AMV RT at 94°C for 2 min and 40 cycles of 94°C for 30 sec, 55°C for 30 sec, and 68°C for 2 min, with a final extension at 68°C for 7 min. RT-PCR was done in 0.2-ml thin-walled PCR tubes (Biozym, Landgraaf, The Netherlands) with a PTC-200 thermocycler (Biozym). Nested PCR was conducted in separate reaction tubes to amplify the 0.46-kb gag3 or 0.7-kb (ES7–ES8) env1 HMA fragments. Two microliters of first-round PCR product of the multiplex RT-PCR was used as template for each nested reaction. Second-round amplifications were carried out in a 100-ml reaction volume.3 HMA was performed as described previously.1,3 On testing with the multiplex RT-PCR assay, 115 of 136 (84.5%) were positive for both gag and env, positive amplifi-
cation for the gag fragment being observed in 130 of 136 (95.6%) samples, while for the env fragment 119 of 136 (87.5%) samples tested positive (Table 1). The difference in sensitivity for the two assays is not clear and may need further investigation. As compared with multiplex RT-PCR, in mono-RT-PCR a separate first-round gag and env amplification is performed under the same reaction conditions as in the multiplex RT-PCR assay. Therefore, the difference in sensitivity may reflect some interference between the multiplexed primer pairs during the RT or the first-round PCR. For the analyzed samples for which viral load data were available, there was no correlation between failure of multiplex RT-PCR assay and low viral load. In a separate study of 30 HIV-1-seropositive individuals from The Gambia we demonstrated that one-tube multiplex PCR also works when the target is not cDNA but proviral DNA from uncultured peripheral blood mononuclear cells (PBMCs). From 27 of 30 (90%) samples both gag and env HMA fragments were amplified simultaneously. In one sample only the gag HMA fragment could be amplified by multiplex DNA PCR, and in two samples amplification was negative for both gag and env HMA in multiplex as well as mono-DNA PCR. 7 Thus when compared with the mono-RT-PCR, one-tube multiplex RT-PCR is cost effective (a 3.5-fold reduction in the price of reagents), less labor-intensive, and highly sensitive. The application of multiplex RT-PCR, which allows generation of HMA fragments for env and gag genes, will be important for the global surveillance of HIV-1 subtypes and recombinants, which will give important information for the development of globally effective HIV vaccines.
ACKNOWLEDGMENTS This work was supported by the Fonds voor Wetenschappelijk Onderzoek, Brussels, Belgium (grant number G.0134.97); the Flanders Interuniversity Institute for Biotechnology (VIB), Ghent, Belgium; and the Human Science Foundation (Tokyo, Japan).
ONE-TUBE MULTIPLEX RT-PCR
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1505 6. Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-van Dillen PME, and van der Noordaa J: Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990;28:495– 503. 7. Cham F, Heyndrickx L, Janssens W, et al.: Study of HIV-1 gag/env variability in The Gambia using a multiplex DNA PCR. AIDS Res Hum Retroviruses 2000;16:(in press).
Address reprint requests to: Wouter Janssens Department of Microbiology Institute of Tropical Medicine Nationalestraat 155 2000 Antwerp, Belgium E-mail:
[email protected] 
