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Updated: Aug 8, 2026
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Development and evaluation of a qualitative reverse-transcriptase nested polymerase chain reaction protocol for
Benoît Lesage1, Anne-Sophie Vannin, Serena Emiliani
1Fertility Clinic, Department of Obstetrics and Gynaecology and Laboratory for Research on Human Reproduction, Campus Erasme, Universite Libre de Bruxelles (ULB), Brussels, Belgium.
Objective:
To develop a method for same-day validation of processed semen in the setting of assisted reproductive techniques (ART) with patients who are seropositive for human immunodeficiency virus, type 1 (HIV-1).
Design:
Laboratory experiments.
Setting:
University hospital.
Patient(S):
Volunteers who are HIV-1 seronegative and seropositive.
Intervention(S):
Evaluation of the sensitivity of a reverse-transcriptase (RT)-nested polymerase chain reaction (PCR) in HIV-1 RNA-positive blood plasma, in artificially infected blood plasma and semen, and in 85 semen samples of 29 HIV-1-seropositive volunteers. Semen was submitted to gradient separation, followed by swim-up.
Main Outcome Measure(S):
Qualitative detection of HIV-1 RNA in blood plasma and in different parts of semen preparation by using RT-nested PCR, PCR inhibition control by dilution of samples, and an internal control.
Result(S):
The detection limit of our PCR was 20 HIV-1 RNA copies per milliliter. Among seropositive patients, RNA was detected in 25% of fresh semen, 36.5% of seminal plasma, 27.5% of gradient supernatants, and 7.1% of final preparations before the migration-sedimentation stage. Positive final preparations were observed in patients who had blood viral loads of >/=20,000 HIV-1 RNA copies per milliliter. Inhibition was present in 17.6% of seminal plasma and in 20% gradient supernatants and in 2 final preparations among 69 tested. Among 25 preparations tested after the migration-sedimentation stage, 2 were positive (1 patient; 70,000 HIV-1 RNA copies per milliliter).
Conclusion(S):
The RT-nested PCR detects low viral load and allows the validation of semen preparations of HIV-1-seropositive patients for ART on the day of sampling. For this purpose, the validation is performed on spermatozoa that are obtained after gradient separation before swim-up. Inhibition of the PCR must be controlled by using an internal control that is well-designed to explore the detection limit of the method.
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