Comparison of MPure-12 Automatic Nucleic Acid Purification and Chelex-100 Method / 法医学杂志

<p><b>OBJECTIVES</b>To explore the forensic application value of MPure-12 automatic nucleic acid purification （MPure-12 Method） for DNA extraction by extracting and typing DNA from bloodstains and various kinds of biological samples with different DNA contents.</p><p><b>METHODS</b>Nine types of biological samples, such as bloodstains, semen stains, and saliva were collected. DNA were extracted using MPure-12 method and Chelex-100 method, followed by PCR amplification and electrophoresis for obtaining STR-profiles.</p><p><b>RESULTS</b>The samples such as hair root, chutty, butt, muscular tissue, saliva stain, bloodstain and semen stain were typed successfully by MPure-12 method. Partial alleles were lacked in the samples of saliva, and the genotyping of contact swabs was unsatisfactory. Additional, all of the bloodstains （20 μL, 15 μL, 10 μL, 5 μL, 1 μL） showed good typing results using Chelex-100 method. But the loss of alleles occurred in 1 μL blood volume by MPure-12 method.</p><p><b>CONCLUSIONS</b>MPure-12 method is suitable for DNA extraction of a certain concentration blood samples．Chelex-100 method may be better for the extraction of trace blood samples．This instrument used in nucleic acid extraction has the advantages of simplicity of operator, rapidity, high extraction efficiency, high rate of reportable STR-profiles and lower man-made pollution.</p>

DNA Quantification of Blood Samples Pre-treated with Pyramidon / 法医学杂志

Objective To study DNA quantification and STR typing of samples pre-treated with pyrami-don. Methods The blood samples of ten unrelated individuals were anticoagulated in EDTA. The blood stains were made on the filter paper. The experimental groups were divided into six groups in accor-dance with the storage time, 30 min, 1 h, 3 h, 6 h, 12 h and 24 h after pre-treated with pyramidon. DNA was extracted by three methods: magnetic bead-based extraction, QIAcube DNA purification method and Chelex-100 method. The quantification of DNA was made by fluorescent quantitative PCR. STR typing was detected by PCR-STR fluorescent technology. Results In the same DNA extraction method, the sample DNA decreased gradually with times after pre-treatment with pyramidon. In the same storage time, the DNA quantification in different extraction methods had significant differences. Sixteen loci DNA typing were detected in 90.56% of samples. Conclusion Pyramidon pre-treatment could cause DNA degradation, but effective STR typing can be achieved within 24 h. The magnetic bead-based extrac-tion is the best method for STR profiling and DNA extraction.

The real-time fluorescence quantitative PCR assay for detection of bacterial contamination in platelet concentrates / 中国输血杂志

Objective To establish a real-time PCR assay to detect bacterial contamination in platelet concentrates(PCs).Methods PCs were spiked with serial dilutions of Staphylococcus aureaus and Eschetichia coli,and the bacterial genomic DNA was isolated by modified Chelex-100 method and then quantitatively detected by Tagman real-time PCR.Filtration was performed to avoid contamination from other bacterial DNA in the reagents of extraction kit as well as the PCR mixture.Results The real-time PCR targeting conservative 16S rRNA gene showed high sensitivity and specificity in detecting bacterial contamination in PCs,and no cross-reaction with human genomic DNA and viruses was found.The Ct value of the lowest bacterial dose groups after filtration in both S.aureaus and E.coli were statistically different from the negative controls,with a minimal detection quality of 0.3CFUs/PCR for S.aureaus,and 0.1CFUs/PCR for E.coli.,respectively.Conclusion The modified Chelex-100 method to extract bacterial genomic DNA and the subsequent real-time PCR are convenient,sensitive and specific,and may be applied to the large scale clinical detection of bacterial contamination in PCs.

Preparation of DNA from Cryptosporidium parvum Oocysts for PCR Detection / 中国寄生虫学与寄生虫病杂志

Objective To establish three methods of DNA extraction from Cryptosporidium parvum oocysts and test by PCR. Methods After three freeze-thaw cycles, three kinds of templates were extracted from the oocysts by Chelex-100, phenol/chloroform or genomic DNA purification system kit, and used for PCR detection. According to the sequence of a C.parvum gene (L16996), a pair of primers was designed and synthesized, and used for PCR. The sensitivity of the template by Chelex-100 method was also tested by PCR. Results One 446 bp PCR product was observed by agarose gel electrophoresis for all three kinds of templates. The PCR sensitivity by Chelex-100 extracted DNA reached for detection of a specimen containing only 1/2 oocyst. Conclusion The three kinds of extraction can all be served as templates for PCR detection of C.parvum oocysts, while Chelex-100 method is simpler, quicker and more reliable for DNA extraction of the parasite.

Comparison on different DNA extraction methods for batches of bloodstain samples during the establishment of national DNA database / 中国法医学杂志

Objective To compare and evaluate different DNA extraction methods for batches of bloodstain samples during the establishment of national DNA database. MethodsDNA was extracted from bloodstain samples collected for the establishment of DNA database using manual Chelex-100, Biomek 3000 automation workstation with Chelex-100 and DNA-IQTM magnetic bead method respectively, and the quantity of DNA was determined by the real-time fluorescent quantitative PCR technique. Genotyping was performed using the AmpFLSTR STR Identifiler kit and 3100 genetic analyzer. ResultsThe concentrations of DNA templates obtained by manual Chelex-100, automatic Chelex-100 and DNA-IQTM magnetic bead method were 0.593?0.131ng/?l, 0.579?0.096ng/?l and 0.447?0.056ng/?l respectively, and the success rates of genotyping with these DNA templates were 100%, 98.9% and 99.5% respectively. ConclusionThe automatic Chelex-100 method proposed by this study can be applied in the establishment of large-scale DNA databases.

Real time PCR quantificational study of DNA extracted by Chelex-100 method / 中国法医学杂志

Objective To study the relation between the quantity of DNA extracted by Chelex-100 method and multiplex STRs analysis.Methods DNA extracted from a variety of common forensic casework specimens were quantified by using Real-time PCR,and then amplified with AmpFLSTR IdentifilerTM PCR Amplification kit.ResultsAccording to the results of quantification,the quantities of DNA extracted from 113 samples by Chelex-100 method were adjusted to 0.5~3ng for establishing 8?l amplification system,and in this condition,most of 113 forensic casework specimens could be successfully genotyped.Conclusion When the quantity of DNA extracted by Chelex-100 method ranged from 0.5ng to 3ng,most results of multiplex STRs analysis were satisfying.Moreover,the amplification effect of 1?l DNA template was better than 3?l DNA template when the concentrations of extracted DNA were more than 0.5ng/?l.