Quantitation of human immunodeficiency virus type 1 DNA by two PCR procedures coupled with enzyme-linked oligosorbent assay

Abstract

Two quantitative PCR methods with our nonisotopic enzyme-linked oligosorbent assay (ELOSA) in microtiter plate format were developed for quantitation of human immunodeficiency virus type 1 (HIV-1). Quantitative competitive PCR (QC-PCR) was based on the coamplification of the wild-type nef region with a mimic competitive nef gene template carrying mutations in the capture region. Correlation of wild-type HIV-1 nef DNA to mimic template copy number permitted quantitation of HIV-1 copy numbers in the range of 20 to 2,000 copies per g of DNA. Internally controlled PCR (IC-PCR) was based on coamplification of the nef region and the ras gene as an internal endogenous standard. Correlation to known amounts of HIV-1 DNA permitted quantitation by IC-PCR of HIV-1 copy numbers in the range of 10 to 2,000 copies per g of DNA. QC-and IC-PCR-ELOSA were performed on a panel of 53 seropositive patients and 12 seronegative controls. The methods showed similar coefficients of variation below 24%. Quantitations by QC-and IC-PCR-ELOSA were identical for 77% of patient samples. The copy level ranged between 443 ؎ 156 and 21,453 ؎ 13,511 copies per 10 5 CD4 cells for asymptomatic and AIDS patients, respectively. The simplicity and reliability of QC-and IC-PCR-ELOSA methods make them appropriate for routine laboratory use in the quantitation of viral and bacterial DNAs.

Clinical Evaluation of an In-House Reverse Transcription-Competitive PCR for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

“…The in-house HIV-1 RNA quantitation procedure described here was directly adapted from a cPCR system previously used for the titration of DNA targets (28). Although presently replaced by the measurement of HIV-1 RNA levels, robust cPCR procedures were initially developed for the quantitation of HIV-1 DNA by several investigators (7,8,13,15). While the routine clinical use of homemade RNA competitors may pose problems in terms of titration and long-term storage, DNA competitors can be easily and reliably titrated and stored indefinitely (3).…”

Comparative Study of Different Standardization Concepts in Quantitative Competitive Reverse Transcription-PCR Assays

“…None of these approaches overcame the problem of inhibition of individual probes. As a consequence, the next generation focused on amplification reactions that were internally controlled, either by coamplification of internal endogenous standards, such as housekeeping genes (5,16), or by introduction of an artificial exogenous mimic fragment (2,9,26). For detailed reviews, see Clementi et al (6,7).…”

“…A greater diversity can be found among standardization concepts. Frequently, a serial-dilution method (referred to here as method A) (Table 1), where either the analyte is diluted and coamplified with a constant amount of internal mimic or vice versa (16,20,22), is applied. Another common standardization format is based on the generation of an external standard curve, where known and increasing amounts of cloned wild-type fragments are coamplified with one constant amount of a mutated competitor mimic (method B) (Table 1).…”

Ποσοτικοποίηση του HIV-DNA σε μονοπύρηνα κύτταρα περιφερικού αίματος (PBMCs) φορέων της HIV λοίμωξης

“…Μηα πξφζθαηε κειέηε, αληίζεηα, απέδεημε φηη ε έλαξμε ιήςεο HAART είρε σο απνηέιεζκα ηελ αχμεζε ησλ ελδνθπηηαξηθψλ επηπέδσλ ησλ κε ελζσκαησκέλσλ κνξθψλ ηνπ HIV-1 DNA212 . Southern Hybridization)220-222, 228, 230, 238-240 , κε ηε βνήζεηα ελδχκσλ184,188,208,224,225,234 , ή ρξεζηκνπνηψληαο βξσκηνχρν αηζίδην226,231 . ζα δηεπθνιχλεη ηε ιήςε απνθάζεσλ ζε αζζελείο πνπ δελ αληαπνθξίλνληαη ζηηο ζεκεξηλέο θαηεπζπληήξηεο νδεγίεο γηα ηελ ζεξαπεία (π.ρ.…”

“…θχηηαξα. Όπσο έρεη αλαθεξζεί απφ αξθεηέο κειέηεο, κεηά απφ έλα ρξφλν απφ ηελ έλαξμε ηεο απνηειεζκαηηθήο αληηξεηξντθήο ζεξαπείαο νη δηαθνξεηηθέο ελδνθπηηαξηθέο κνξθέο ηνπ ηνχ HIV-1 DNA είλαη ζεκαληηθά θαηαζηαικέλεο[198][199][200][201][202][203][204][205][206][207][208][209][210][211] , ζε αληίζεζε κε ηνπο αζζελείο πνπ 2.10.2.10.3.1 Γνθηκαζίεο βαζηζκέλεο ζηελ ηερλνινγία ηεο ζπκβαηηθήο PCR.Έρνπλ αλαπηπρζεί πνιπάξηζκεο δνθηκαζίεο πνπ βαζίδνληαη ζηελ ηερλνινγία ηεο ζπκβαηηθήο PCR γηα ηελ πνζνηηθνπνίεζε ησλ επηπέδσλ ηνπ ελδνθπηηαξηθνχ HIV-1 DNA, θαηά ηηο νπνίεο ε πνζνηηθνπνίεζε ηνπ πξντφληνο ηεο αληίδξαζεο πξαγκαηνπνηείηαη κεηά ην ηέινο ηεο184,188,199,208,209,[218][219][220][221][222][223][224][225][226][227][228][229][230][231][232][233][234][235] . Ο πνζνηηθφο πξνζδηνξηζκφο ησλ πξντφλησλ ηεο αληίδξαζεο PCR ζε φιεο απηέο ηηο κεζφδνπο κε ρξήζε ζπκβαηηθήο ηερινγίαο PCR επηηπγράλεηαη κε ηε ζχγθξηζε ηεο ηζρχνο ηνπ ζήκαηνο απφ ηα πξφηππα ηεο αληίδξαζεο (εμσηεξηθά ή / θαη εζσηεξηθά) κε γλσζηέο ζπγθεληξψζεηο, ηα νπνία έρνπλ πνζνηηθνπνηεζεί ηελ ίδηα ζηηγκή θαη ηα άγλσζηα, ππφ πνζνηηθνπνίεζε δείγκαηα.…”