DEVELOPMENT OF A MULTIPLEX PCR ASSAY FOR THE SIMULTANEOUS DETECTION AND DISCRIMINATION OF HIV-1, HIV-2, HTLV-I AND HTLV-II

Background: Multiplex polymerase chain reaction (PCR) has been establi shed as a general technique for the simultaneous amplification of diff erent target sequences. Uses of multiplex include pathogens identifica tion, linkage analysis and genetic disease diagnosis. The high sensiti vity of PCR may produce false-positive results due to contamination wi th previously amplified material. Objectives: To develop a multiplex P CR technique that can simultaneously detect and discriminate human imm unodeficiency virus types 1 and 2 (HIV-1/2) and human T-lymphotropic v irus types 1 and 2 (HTLV-I/II) proviral sequences. Such a method shoul d incorporate a system that prevents the occurrence of false-positive results. Study design: Combinations of four primer pairs, one for each retrovirus, were assayed in order to determine the combination of oli gonucleotides as well as the PCR conditions that yield the most specif ic and sensitive coamplification of proviral sequences. To prevent con tamination with DNA from previous PCR amplifications, the uracil N-gly cosylase (UNG) system was incorporated into the coamplification format . Results. A combination of primer pairs from the gag region of HIV-1, env of HIV-2, pol of HTLV-I and tax of HTLV-II yielded specific and s ensitive coamplification of proviral sequences. The UNG system was inc orporated and shown to be efficient in the degradation of contaminatin g DNA. In the evaluation of a serologically well established panel of singly and dually infected individuals, the assay detected 20/22 HIV-1 , 8/10 HIV-2, 8/8 HTLV-I and 8/8 HTLV-II infections. Conclusions: A mu ltiplex PCR method for the detection and discrimination of HIV-1/2 and HTLV-I/II has been developed. Under standardized conditions, all four proviral sequences were detected in a specific and sensitive manner. The evaluation of a panel of clinical specimens from infected individu als by one or more retroviruses showed that the technique detected mos t of the infected individuals. A low viral load may explain cases wher e multiplex PCR failed to detect target sequences. (C) 1996 Elsevier S cience B.V.