TY - JOUR
T1 - Recovery and analysis of human immunodeficiency virus type 1 (HIV) RNA sequences from plasma samples with low HIV RNA levels
AU - Niubò, Jordi
AU - Li, Wuyi
AU - Henry, Keith
AU - Erice, Alejo
PY - 2000
Y1 - 2000
N2 - Amplification of human immunodeficiency virus type 1 (HIV) reverse transcriptase (RT) and protease (PT) sequences from plasma is difficult when HIV RNA levels are low, and it usually cannot be accomplished in samples with <1,000 HIV RNA copies/ml. Because the RNA extraction step is critical for the success of subsequent amplifications and sequence analyses, two RNA extraction methods were compared to study plasma samples with low HIV RNA levels. Forty-four plasma samples containing <500 HIV RNA copies/ml in a branched-DNA (bDNA) assay (Quantiplex HIV RNA assay version 2.0 [Chiron Corp., Emeryville, Calif.]) were studied. RNA was extracted by using two commercial kits (QIAamp Viral RNA kit [Qiagen, Hilden, Germany] and NucliSens kit [Organon Teknika, Boxtel, The Netherlands]). Fragments (1,144 bp) encompassing HIV PT and RT sequences were amplified by nested PCRs. Amplified products were sequenced by using a commercial kit (Applied Biosystems). HIV RNA was recovered from a total of 21 plasma samples, including 20 samples after extraction by the NucliSens method, and 8 samples after extraction by the QIAamp method (P < 0.05). Mean HIV RNA levels in these samples, measured by an ultrasensitive bDNA assay (Quantiplex HIV RNA assay version 3.0; Chiron Corp., Emeryville, Calif.), were 848 copies/ml (median, 666; range, 154 to 2,606 copies/ml). Analysis of RT and PT sequences in five samples demonstrated an average of 3.8 and 2.4 resistance mutations in these regions, respectively. The NucliSens RNA extraction kit is a valuable method for obtaining HIV RNA for genotypic studies from plasma fractions of individuals with low HIV RNA levels.
AB - Amplification of human immunodeficiency virus type 1 (HIV) reverse transcriptase (RT) and protease (PT) sequences from plasma is difficult when HIV RNA levels are low, and it usually cannot be accomplished in samples with <1,000 HIV RNA copies/ml. Because the RNA extraction step is critical for the success of subsequent amplifications and sequence analyses, two RNA extraction methods were compared to study plasma samples with low HIV RNA levels. Forty-four plasma samples containing <500 HIV RNA copies/ml in a branched-DNA (bDNA) assay (Quantiplex HIV RNA assay version 2.0 [Chiron Corp., Emeryville, Calif.]) were studied. RNA was extracted by using two commercial kits (QIAamp Viral RNA kit [Qiagen, Hilden, Germany] and NucliSens kit [Organon Teknika, Boxtel, The Netherlands]). Fragments (1,144 bp) encompassing HIV PT and RT sequences were amplified by nested PCRs. Amplified products were sequenced by using a commercial kit (Applied Biosystems). HIV RNA was recovered from a total of 21 plasma samples, including 20 samples after extraction by the NucliSens method, and 8 samples after extraction by the QIAamp method (P < 0.05). Mean HIV RNA levels in these samples, measured by an ultrasensitive bDNA assay (Quantiplex HIV RNA assay version 3.0; Chiron Corp., Emeryville, Calif.), were 848 copies/ml (median, 666; range, 154 to 2,606 copies/ml). Analysis of RT and PT sequences in five samples demonstrated an average of 3.8 and 2.4 resistance mutations in these regions, respectively. The NucliSens RNA extraction kit is a valuable method for obtaining HIV RNA for genotypic studies from plasma fractions of individuals with low HIV RNA levels.
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M3 - Article
C2 - 10618106
AN - SCOPUS:0033989441
SN - 0095-1137
VL - 38
SP - 309
EP - 312
JO - Journal of clinical microbiology
JF - Journal of clinical microbiology
IS - 1
ER -