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.


OBJECTIVE
To develop a multiplex PCR technique that can simultaneously detect and discriminate human immunodeficiency virus types 1 and 2 (HIV-1/2) and human T-lymphotropic virus types 1 and 2 (HTLV-I/II) proviral sequences. Such a method should incorporate a system that prevents the occurrence of false-positive results.


METHODS
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 20/22 HIV-1, 8/10 HIV-2, 8/8 HTLV-I and 8/8 HTLV-II infections.
Heredia
A
A
Laboratory of Molecular Virology, Food and Drug Administration, Rockville, MD 20852-1448, USA.
Soriano
V
V
Weiss
S H
SH
Bravo
R
R
Vallejo
A
A
Denny
T N
TN
Epstein
J S
JS
Hewlett
I K
IK
D002460:Cell Line; D002461:Cell Line, Transformed; D004279:DNA, Viral / Q000032:analysis; D015658:HIV Infections / Q000097:blood / Q000821:virology; D015497:HIV-1 / Q000302:isolation & purification*; D015498:HIV-2 / Q000302:isolation & purification*; D015490:HTLV-I Infections / Q000097:blood / Q000821:virology; D015491:HTLV-II Infections / Q000097:blood / Q000821:virology; D015368:Human T-lymphotropic virus 1 / Q000302:isolation & purification*; D015367:Human T-lymphotropic virus 2 / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
D004279:DNA, Viral