Quantitation of HIV-1 RNA in breast milk by real time PCR.
HIV-1 RNA in breast milk is a strong predictor of HIV-1 transmission through breastfeeding. In the present report, breast milk samples from HIV-1 uninfected donors were spiked with dilution of quantified culture supernatant from HIV-1(NDK) infected PBMC. Two RNA extraction techniques based on silica extraction, Nuclisens (BioMerieux) and Triazol (Qiagen), two techniques based on guanidine thiocynanate/chloroforme extraction, TRIzol (Life Technologie) and Amplicor HIV-1 Monitor (Roche Diagnostic Systems), and one technique based on electrostatic adsorption on iron oxide micro beads (Promega) were compared. HIV-1 RNA was quantitated by real time PCR (LTR gene) and Amplicor HIV-1 Monitor. Combining magnetic micro beads extraction and real time PCR quantitation allowed to correctly quantify breast milk HIV-1 RNA, with a difference between the expected and measured HIV-1 RNA levels always lower than 0.3 log copies/ml. The same combination was confirmed on 25 breast milk samples from HIV-1 infected women collected in Kwazulu-Natal, South Africa, by comparing measurements with those obtained by the Amplicor HIV-1 Monitor (r(2)=0.88). Nucleic acid extraction by magnetic micro beads followed by real time PCR is a reliable, sensitive, rapid and simple procedure to quantify HIV-1 RNA in breast milk and allows for PCR inhibitors found frequently in these samples.
Becquart
Pierre
P
Research Unit 145, Institute for Research and Development, University of Montpellier 1, France.
Foulongne
Vincent
V
Willumsen
Juana
J
Rouzioux
Christine
C
Segondy
Michel
M
Van de Perre
Philippe
P
D005260:Female; D018628:Gene Dosage; D015658:HIV Infections / Q000821:virology*; D018023:HIV Seronegativity; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D008280:Magnetics; D008863:Microspheres; D008895:Milk, Human / Q000821:virology*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic