Optimization of polymerase chain reaction for detection of HIV type 2 DNA.
The aim of this study was to increase the sensitivity of an earlier version of an HIV-2 nested PCR assay based on primers in the gag, pol, LTR, and env regions. The assay was first optimized with regard to concentrations of dNTP, MgCl2, and primers, using a method that allowed optimization of all three parameters in only two test runs. We then designed and optimized new primer sets in the LTR, gag, and gag/pol regions that were based on more isolates than were the former (old) primer sets. Samples from 57 HIV-2 antibody-positive individuals were tested with the four old primer sets as well as with the three new primer sets. Five primer sets from this run (new gag, new gag/pol, old LTR, old env, and new LTR) were then tested with 35 more samples, giving a total number of 92 tested samples from HIV-2-infected individuals. At initial testing of the 92 samples a combination of primer sets from two different regions yielded a sensitivity ranging from 93.5 to 98.9%. After repeated testing the sensitivity ranged from 96.7 to 100% for the different primer combinations. The specificity was 100% for all primer sets except old LTR, which had a specificity of 97%. In conclusion, it is possible to create a more sensitive PCR assay by optimizing the different PCR parameters as well as by including primer sets based on more isolates.
Walther
L
L
Swedish Institute for Infectious Disease Control and Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm.
Grankvist
O
O
Mirzai
B
B
da Silva
Z
Z
Fredlund
H
H
Biberfeld
G
G
Thorstensson
R
R
D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis*; D015683:Gene Products, gag; D015658:HIV Infections / Q000821:virology; D016325:HIV Long Terminal Repeat; D006679:HIV Seropositivity / Q000821:virology*; D015498:HIV-2 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral; D015683:Gene Products, gag