ABSTRACT
Abstract Molecular tools have improved conventional veterinary diagnosis. Acid nucleic extraction is a key step for downstream applications. This work aimed to compare the DNA extraction method Chelex-100 resin (M1) with Whatman® cards (M2), phenol-chloroform (M3), or commercial kits (M4), and to determine the most sensitive and inexpensive one for its diagnosis of animal pathogens that, despite their economic or zoonotic relevance, receive little attention. DNA was isolated from urine, organs, semen, blood and intestinal mucous, from the bacteria Leptospira interrogans serovar Pomona Pomona (by M1 and M2), Brucella melitensis (by M1, M3 and M4), and Salmonella ser. Abortusequi (by M1 and M4), and the parasites Leishmania spp. (by M1, M3 and M4), and Eimeria spp. (by M1 and M3), respectively. The sensitivity of each method was assayed by Polymerase Chain Reaction (PCR). The M1 showed similar sensitivity for Salmonella ser. Abortusequi, Leishmania spp., and Eimeria spp., being better for L. interrogans serovar Pomona Pomona and slightly lower for B. melitensis. For the first time, a simple and economic method was successfully employed for extracting DNA from these animal pathogens, especially important in low-resource settings, contributing to the diagnosis of leptospirosis, brucellosis, leishmaniasis, and coccidiosis; as well as to the molecular epidemiology of salmonellosis in stallion from semen samples.
Resumen Las técnicas moleculares han contribuido a mejorar el diagnóstico veterinario tradicional y la extracción de ácidos nucleicos es determinante. El objetivo de este trabajo fue comparar el método de extracción de ADN Chelex-100 (M1) con papel Whatman (M2), fenol-cloroformo (M3) o kits comerciales (M4), y determinar un método sensible y de bajo costo para el diagnóstico de patógenos de animales económica o zoonóticamente relevantes y que reciben poca atención. A partir de orina, órganos, semen, sangre y mucosa intestinal se extrajo el ADN de las bacterias Leptospira interrogans serovar Pomona Pomona (con M1 y M2), Brucella melitensis (con M1, M3 y M4), Salmonella ser. Abortusequi (M1 y M4), y de los parásitos Leishmania spp. (M1, M3 y M4) y Eimeria spp. (M1 y M3), respectivamente. La sensibilidad de los protocolos fue analizada por PCR. El método M1 demostró una sensibilidad similar para S. Abortusequi, Leishmania spp. y Eimeria spp., siendo mejor para L. interrogans y levemente menor para B. melitensis. Por primera vez se usó exitosamente en estos patógenos veterinarios un método simple y económico para extraer ADN, especialmente importante en laboratorios de bajos recursos económicos, contribuyendo al diagnóstico de leptospirosis, brucelosis, leishmaniasis y coccidiosis, así como también a la epidemiología molecular de salmonelosis en muestras de semen de caballos.
ABSTRACT
<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>
Subject(s)
Humans , Male , Alleles , Blood Stains , Chelating Agents , DNA/isolation & purification , DNA Fingerprinting , Forensic Medicine/methods , Genotype , Polymerase Chain Reaction/methods , Polystyrenes , Polyvinyls , Resins, Synthetic , Saliva , Semen/chemistryABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
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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.