ABSTRACT
Objective To evaluate the efficiency of REPLI-g® Single Cell Kit for sample DNA amplification, and explore its application value in forensic trace DNA amplification. Methods Three DNA extraction kits were selected to extract DNA from peripheral blood of 10 unrelated individuals. The DNA yield and purity of the three DNA extraction kits were compared. According to the results of comparison, one DNA sample was selected to concentrate and dilute, then used as the initial sample of whole genome amplification （WGA）. REPLI-g® Single Cell Kit was used to amplify the initial sample at the whole genome level. The amplification yield and amplification times were calculated, and the distribution of DNA fragments was detected by agarose gel electrophoresis. Goldeneye® DNA ID System 20A Kit was used to perform the STR typing of the initial sample and DNA samples amplified at the whole genome level to evaluate the performance of REPLI-g® Single Cell Kit in trace DNA amplication in terms of purity and yield as well as the success rate of STR typing. Results After comparison, one DNA sample was selected from QIAsymphony® DNA Investigator® Kit extracts to concentrate and dilute as the initial sample of WGA. After amplifying the whole genome of a series of initial samples by REPLI-g® Single Cell Kit, the lowest average of amplification yield reached 8.77×103 ng, while the average of the corresponding amplification times reached 1.40×106. DNA fragments were large and concentrated. The STR typing success rate of WGA samples became lower with the decrease of initial samples used, but when the amount of samples was lower than 0.5 ng, the STR typing success rate of samples after DNA WGA was higher than that of samples without DNA WGA. Conclusion REPLI-g® Single Cell Kit can increase the yield of template DNA. Especially for trace DNA, the STR typing success rate can be improved to a certain extent.
Subject(s)
Humans , DNA , DNA Fingerprinting , Microsatellite Repeats , Nucleic Acid Amplification Techniques/standards , Sequence Analysis, DNA/methodsABSTRACT
BACKGROUND & OBJECTIVE: India has a high prevalence of HIV-1, hapatitis C and B virus (HCV and HBV) in the blood donors but has yet to implement nucleic acid testing (NAT) in blood screening. We undertook a multicentre evaluation of blood donor testing by NAT for simultaneous detection of HIV-1, HBV and HCV in a single tube and also to determine the feasibility of NAT implementation in India's low volume setting. METHODS: A total of 12,224 unlinked samples along with their serological results were obtained from representative eight blood banks in India and were individually manually tested by the Procleix Ultrio Assay (Chiron Corp. Emeryville, CA) for simultaneous detection of HIV-1, HCV, and HBV. RESULTS: Of the 12,224 samples tested, 209 (1.71%) were seroreactive. One hundred thirty three samples (1.09%) were reactive by Ultrio assay, 84 samples were seroreactive but NAT non reactive. There were eight NAT yield cases: 1 HIV, 1 HIV-HCV co-infection, and 6 HBV. INTERPRETATION & CONCLUSION: Our observed NAT yield for all three viruses was 1 in 1528 (0.065%). We estimate NAT could interdict 3272 infectious donations a year among our approximate 5 million annual donations.