Evaluating the Efficiency of REPLI-g® Single Cell Kit for Trace DNA Amplification / 法医学杂志

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.

Determination of cotinine in human urine with gas chromatograph-mass spectrometry / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To establish the method of gas chromatograph-mass spectrometry (GC-MS) for the determination of the cotinine (COT) in human urine.</p><p><b>METHODS</b>The conjugated trans-3'-hydroxycotinine (THOC) and COT were hydrolyzed in human urine with beta-glucuronidase. The composition of COT was extracted with the mixture of dichloromethane and n-butyl acetate (2:1) and was separated with HP-5MS fused-silica capillary column. The GC-MS was used for determining its content.</p><p><b>RESULTS</b>The monitoring limit of this method was 0.02 microg/L. Its recovery rate was higher than 90%, Its accuracy rate was 4.30%. It was used for the determination of the cotinine in human urine in Guangzhou Biological Bank the Elderly Cohort.</p><p><b>CONCLUSION</b>The GC-MS method is a good microanalysis for monitoring the cotinine in human urine rapidly and accurately with little background disturbance. It has been applied in our Guangzhou Cohort Study for determining cotinine in human urine.</p>