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 established as a general technique for the simultaneous amplification of different target sequences. Uses of multiplex include pathogens identification, linkage analysis and genetic disease diagnosis. The high sensitivity of PCR may produce false-positive results due to contamination with previously amplified material.

Objectives: To develop a multiplex PCR technique that can simultaneously detect and discriminate human immunodeficiency virus types 1 and 2 ($\text{HIV}\text{-1}\text{2}$) and human T-lymphotropic virus types 1 and 2 ($\text{HTLV-I}\text{II}$) proviral sequences. Such a method should 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 oligonucleotides as well as the PCR conditions that yield the most specific and sensitive coamplification of proviral sequences. To prevent contamination with DNA from previous PCR amplifications, the uracil N-glycosylase (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 sensitive coamplification of proviral sequences. The UNG system was incorporated and shown to be efficient in the degradation of contaminating DNA. In the evaluation of a serologically well established panel of singly and dually infected individuals, the assay detected $\text{20}\text{22}\text{HIV-1}$, $\text{8}\text{10}\text{HIV-2}$, $\text{8}\text{8}\text{HTLV-I}$ and $\text{8}\text{8}\text{HTLV-II}$ infections.

Conclusions: A multiplex PCR method for the detection and discrimination of $\text{HIV}\text{-1}\text{2}$ and $\text{HTLV-I}\text{II}$ 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 individuals by one or more retroviruses showed that the technique detected most of the infected individuals. A low viral load may explain cases where multiplex PCR failed to detect target sequences.