为实现食品中沙门氏菌的简便和快速现场检测,本研究采用FTA膜(Flinders technology associates,FTA)结合跨越式滚环等温扩增(Saltatory rolling circle amplification,SRCA)方法(FTA-SRCA)建立一种新型的沙门氏菌检测方法。利用FTA膜快...为实现食品中沙门氏菌的简便和快速现场检测,本研究采用FTA膜(Flinders technology associates,FTA)结合跨越式滚环等温扩增(Saltatory rolling circle amplification,SRCA)方法(FTA-SRCA)建立一种新型的沙门氏菌检测方法。利用FTA膜快速提取模板DNA,根据沙门氏菌的inv A基因设计及筛选引物,建立FTA-SRCA反应体系。扩增反应在能够实现集约化检测的凹孔板中进行,反应结束后添加荧光染料观察结果。确定了该方法的特异性、灵敏度和人工污染样品的检出限,并对60个实际样品进行检测,评估其敏感性、特异性和符合率。结果表明:检测的17株沙门氏菌均为阳性结果,29株非沙门氏菌均为阴性结果,特异性良好。FTA-SRCA方法的灵敏度为6.81×100 CFU/m L,比PCR方法高100倍,比SRCA方法高10倍。对于人工污染的牛奶样品检测,FTA-SRCA方法的检出限为3.22×100CFU/m L,比PCR方法低1000倍,比SRCA方法低10倍。检测实际样品的敏感性、特异性和符合率分别为100.00%,94.64%,95.00%。本研究建立的FTA-SRCA方法具有操作简便快速、成本低廉、特异性强、灵敏度高、检出限低等优点,可用于食品中沙门氏菌的大批量集约化快速现场检测。展开更多
The extraction of DNA for molecular analyses is often a limiting factor in terms of cost, time, and availability of skilled labor Here the authors describe a simple extraction protocol for obtaining DNA from field and...The extraction of DNA for molecular analyses is often a limiting factor in terms of cost, time, and availability of skilled labor Here the authors describe a simple extraction protocol for obtaining DNA from field and greenhouse grown plants. This method was optimized for sampling mature wild sorghum populations in remote areas of Ethiopia and young greenhouse grown seedlings for genetic studies using microsatellite loci. Initially, the leaf squashes are made on Whatman FTA plant saver cards or small cards of Whatman chromatography paper. After several washes, sufficient DNA to run up to 70 PCR amplifications is eluted from a 6 mm disk. Both types of cards were equally effective for collecting genomic DNA from young and mature sorghum plants for PCR-based analyses. The use of this technology for extracting genomic DNA from seedlings and/or mature plants in situ is particularly attractive for sampling at sites that are far from the laboratory where samples are ultimately analyzed. Moreover, highly skilled personnel are not required to collect DNA samples using this protocol. The Whatman FTA card is more expensive than the Wbatman chromatography paper. Therefore, without compromising efficiency, the lower cost chromatography paper can be used for DNA extraction, especially for institutions in developing countries.展开更多
Background: The incidence of mycobacterial infection, in particular M. tuberculosis complex (MTC), is increasing in some Western countries, while nontuberculous mycobacteria (NTM) may be recognized more frequently in ...Background: The incidence of mycobacterial infection, in particular M. tuberculosis complex (MTC), is increasing in some Western countries, while nontuberculous mycobacteria (NTM) may be recognized more frequently in clinical specimens worldwide. The clinical scenario and available histopathology alone are often insufficient to separate these two categories of mycobacterial disease, whose behavior and treatment differ. In particular, NTM may be clinically unsuspected in pathological specimens and the opportunity for culturing missed. Methods: We developed two multiplex PCR assays, which distinguish MTC from NTM by detecting the IS6110 insert in the first tube and discriminating up to 14 NTM reference strains in the second by targeting the 16S-23S rRNA internal transcribed spacer. Test material included 594 routine clinical specimens with diverse pathology;many were granulomas unrelated to mycobacterial infection. About 75% were formalin-fixed paraffin blocks, the remainder mainly cytologic imprints or aspirates on FTA cards submitted on suspicion of mycobacterial infection either to avoid frozen sectioning (with the attendant risk of aerosolisation) or at the time of fine needle aspiration. Results: The paraffinized material yielded 53 MTC positives and the cytological 21 positives. A subset consisting of 337 specimens was also analyzed for NTM and yielded 51 positives. The frequency of simultaneous NTM infection in tuberculous patients was about 17%. Mycobacterium avium complex represented the dominant NTM species overall, showed a predilection for lung and lymph node, and together with M. haemophilum were the second most frequent NTM just behind M. ulcerans/M. marinum in skin and soft tissue, the category displaying the largest NTM diversity. Conclusions: Cytological and deparaffinized tissue analyzed in a new two-tube multiplex PCR allows for specific discrimination of causative agents in mycobacterial infection. MTC is readily distinguished from NTM for appropriate therapy, and NTM presumptively diagnosed at the species level allows appropriate choices of antimicrobials.展开更多
This study evaluated the performance of the Wuxi AGCU ScienTech Incorporation(HuiShan,Wuxi,China)AGCU Expressmarker 16(EX 16)and 22(EX22)short tandem repeat(STR)amplification kits in reduced reaction volumes using dir...This study evaluated the performance of the Wuxi AGCU ScienTech Incorporation(HuiShan,Wuxi,China)AGCU Expressmarker 16(EX 16)and 22(EX22)short tandem repeat(STR)amplification kits in reduced reaction volumes using direct polymerase chain reaction(PCR)amplification workflows.The commercially available PowerPlex21(PP21)System(Promega,Wisconsin,USA),which follows similar direct workflows,was used as a reference.Anticoagulate blood applied to chemically impregnated FTATM Micro Cards(GE Healthcare UK Limited,Amersham Place,Little Chalfont,Buckinghamshire,HP79NA,UK)was used to represent a complex biological sample.Allelic concordance,first‑pass success rate,average peak heights,heterozygous peak height ratios(HPHRs),and intracolor and intercolor peak height balance were determined.In reduced volume PCR reactions,the performances of both the EX16 and EX22 STR amplification kits were comparable to that of the PP21 System.The level of performance was maintained at PCR reaction volumes,which are 40%of that recommended.The EX22 and PP21 System kits possess comparable overlapping genome coverage.This study evaluated the performance of the AGCU EX16 and EX22 STR amplification kits in reduced PCR reaction volumes using direct workflows in combination with whole blood applied to FTATM Micro Cards.Allelic concordance,first‑pass success rate,average peak heights,HPHRs,and intracolor and intercolor peak height balance were determined.A concordance analysis was completed that compared the performance of the EX16 and EX22 kits using human blood applied to FTA Micro Cards in combination with full,half,and reduced PCR reaction volumes.The PP21 System(Promega)was used as a reference kit.Where appropriate,the distributions of data were assessed using the Shapiro‑Wilk test.For normally‑distributed data,statistics were calculated using analysis of variance(ANOVA)and for nonparametric data the Wilcoxon/Kruskal‑Wallis test was used.Statistical significance was set at P<0.05.Confidence intervals for mean values were set at 95%.On using reduced volume PCR reactions in combination with dried blood spots applied to FTA sample collection cards,both the EX16 and EX22 kits were shown to generate STR profiles of sufficient quality to allow entry into National DNA databases.The performance of both EX16 and EX22 was comparable to that of the PP21 System.This study demonstrates the successful use of the Wuxi AGCU ScienTech Incorporation EX16 and EX22 kits in reduced PCR reaction volumes with complex biological samples applied to chemically impregnated FTA sample collection cards.展开更多
Regulatory Standards and Forensic Communities are expressing an expectation for HID products to be certified as“DNA‑free.”Recently,“DNA‑free”status was described for HID‑related products using ethylene oxide(EtO);...Regulatory Standards and Forensic Communities are expressing an expectation for HID products to be certified as“DNA‑free.”Recently,“DNA‑free”status was described for HID‑related products using ethylene oxide(EtO);this gas reduces the presence of amplifiable DNA and causes minimal interference to downstream HID‑analytical methods.During sample collection,indicating cards,for example,Indicating FTA™(GE Healthcare Life Sciences,UK),are used to collect and store buccal cell DNA.These cards contain a dye which changes color on application of a colorless sample.Generating“DNA‑free”indicating cards using EtO should not impact the dyes’ability to indicate sample location or the efficacy of the card in downstream HID‑analytical methods.This study was initiated to identify alternative dyes to those currently used with sample indicating collection cards.The most promising,dyes when applied to cellulose papers exhibited a uniform color distribution and excellent sample indicating properties even when mixed with chemicals associated with FTA™.When dyed cellulose papers were exposed to EtO,ultraviolet radiation,elevated temperature,and humidity,negligible fading or discoloration was observed.The presence of these dyes on cellulose papers did not interfere with direct short tandem repeat(STR)profiling.Allelic concordance,first pass success rate,and mean peak heights were comparable to samples applied to Indicating FTA.Biological samples applied to EtO‑treated dyed cellulose papers and stored>1 month produced full STR profiles of sufficient quality to allow submission to DNA databases,confirming negligible interference from EtO treatment.These alternative sample indicating dyes resist EtO‑mediated fading while fulfilling the Forensic Community’s expectation for“DNA‑free”with negligible impact on collection card performance.展开更多
文摘为实现食品中沙门氏菌的简便和快速现场检测,本研究采用FTA膜(Flinders technology associates,FTA)结合跨越式滚环等温扩增(Saltatory rolling circle amplification,SRCA)方法(FTA-SRCA)建立一种新型的沙门氏菌检测方法。利用FTA膜快速提取模板DNA,根据沙门氏菌的inv A基因设计及筛选引物,建立FTA-SRCA反应体系。扩增反应在能够实现集约化检测的凹孔板中进行,反应结束后添加荧光染料观察结果。确定了该方法的特异性、灵敏度和人工污染样品的检出限,并对60个实际样品进行检测,评估其敏感性、特异性和符合率。结果表明:检测的17株沙门氏菌均为阳性结果,29株非沙门氏菌均为阴性结果,特异性良好。FTA-SRCA方法的灵敏度为6.81×100 CFU/m L,比PCR方法高100倍,比SRCA方法高10倍。对于人工污染的牛奶样品检测,FTA-SRCA方法的检出限为3.22×100CFU/m L,比PCR方法低1000倍,比SRCA方法低10倍。检测实际样品的敏感性、特异性和符合率分别为100.00%,94.64%,95.00%。本研究建立的FTA-SRCA方法具有操作简便快速、成本低廉、特异性强、灵敏度高、检出限低等优点,可用于食品中沙门氏菌的大批量集约化快速现场检测。
文摘The extraction of DNA for molecular analyses is often a limiting factor in terms of cost, time, and availability of skilled labor Here the authors describe a simple extraction protocol for obtaining DNA from field and greenhouse grown plants. This method was optimized for sampling mature wild sorghum populations in remote areas of Ethiopia and young greenhouse grown seedlings for genetic studies using microsatellite loci. Initially, the leaf squashes are made on Whatman FTA plant saver cards or small cards of Whatman chromatography paper. After several washes, sufficient DNA to run up to 70 PCR amplifications is eluted from a 6 mm disk. Both types of cards were equally effective for collecting genomic DNA from young and mature sorghum plants for PCR-based analyses. The use of this technology for extracting genomic DNA from seedlings and/or mature plants in situ is particularly attractive for sampling at sites that are far from the laboratory where samples are ultimately analyzed. Moreover, highly skilled personnel are not required to collect DNA samples using this protocol. The Whatman FTA card is more expensive than the Wbatman chromatography paper. Therefore, without compromising efficiency, the lower cost chromatography paper can be used for DNA extraction, especially for institutions in developing countries.
文摘Background: The incidence of mycobacterial infection, in particular M. tuberculosis complex (MTC), is increasing in some Western countries, while nontuberculous mycobacteria (NTM) may be recognized more frequently in clinical specimens worldwide. The clinical scenario and available histopathology alone are often insufficient to separate these two categories of mycobacterial disease, whose behavior and treatment differ. In particular, NTM may be clinically unsuspected in pathological specimens and the opportunity for culturing missed. Methods: We developed two multiplex PCR assays, which distinguish MTC from NTM by detecting the IS6110 insert in the first tube and discriminating up to 14 NTM reference strains in the second by targeting the 16S-23S rRNA internal transcribed spacer. Test material included 594 routine clinical specimens with diverse pathology;many were granulomas unrelated to mycobacterial infection. About 75% were formalin-fixed paraffin blocks, the remainder mainly cytologic imprints or aspirates on FTA cards submitted on suspicion of mycobacterial infection either to avoid frozen sectioning (with the attendant risk of aerosolisation) or at the time of fine needle aspiration. Results: The paraffinized material yielded 53 MTC positives and the cytological 21 positives. A subset consisting of 337 specimens was also analyzed for NTM and yielded 51 positives. The frequency of simultaneous NTM infection in tuberculous patients was about 17%. Mycobacterium avium complex represented the dominant NTM species overall, showed a predilection for lung and lymph node, and together with M. haemophilum were the second most frequent NTM just behind M. ulcerans/M. marinum in skin and soft tissue, the category displaying the largest NTM diversity. Conclusions: Cytological and deparaffinized tissue analyzed in a new two-tube multiplex PCR allows for specific discrimination of causative agents in mycobacterial infection. MTC is readily distinguished from NTM for appropriate therapy, and NTM presumptively diagnosed at the species level allows appropriate choices of antimicrobials.
文摘This study evaluated the performance of the Wuxi AGCU ScienTech Incorporation(HuiShan,Wuxi,China)AGCU Expressmarker 16(EX 16)and 22(EX22)short tandem repeat(STR)amplification kits in reduced reaction volumes using direct polymerase chain reaction(PCR)amplification workflows.The commercially available PowerPlex21(PP21)System(Promega,Wisconsin,USA),which follows similar direct workflows,was used as a reference.Anticoagulate blood applied to chemically impregnated FTATM Micro Cards(GE Healthcare UK Limited,Amersham Place,Little Chalfont,Buckinghamshire,HP79NA,UK)was used to represent a complex biological sample.Allelic concordance,first‑pass success rate,average peak heights,heterozygous peak height ratios(HPHRs),and intracolor and intercolor peak height balance were determined.In reduced volume PCR reactions,the performances of both the EX16 and EX22 STR amplification kits were comparable to that of the PP21 System.The level of performance was maintained at PCR reaction volumes,which are 40%of that recommended.The EX22 and PP21 System kits possess comparable overlapping genome coverage.This study evaluated the performance of the AGCU EX16 and EX22 STR amplification kits in reduced PCR reaction volumes using direct workflows in combination with whole blood applied to FTATM Micro Cards.Allelic concordance,first‑pass success rate,average peak heights,HPHRs,and intracolor and intercolor peak height balance were determined.A concordance analysis was completed that compared the performance of the EX16 and EX22 kits using human blood applied to FTA Micro Cards in combination with full,half,and reduced PCR reaction volumes.The PP21 System(Promega)was used as a reference kit.Where appropriate,the distributions of data were assessed using the Shapiro‑Wilk test.For normally‑distributed data,statistics were calculated using analysis of variance(ANOVA)and for nonparametric data the Wilcoxon/Kruskal‑Wallis test was used.Statistical significance was set at P<0.05.Confidence intervals for mean values were set at 95%.On using reduced volume PCR reactions in combination with dried blood spots applied to FTA sample collection cards,both the EX16 and EX22 kits were shown to generate STR profiles of sufficient quality to allow entry into National DNA databases.The performance of both EX16 and EX22 was comparable to that of the PP21 System.This study demonstrates the successful use of the Wuxi AGCU ScienTech Incorporation EX16 and EX22 kits in reduced PCR reaction volumes with complex biological samples applied to chemically impregnated FTA sample collection cards.
文摘Regulatory Standards and Forensic Communities are expressing an expectation for HID products to be certified as“DNA‑free.”Recently,“DNA‑free”status was described for HID‑related products using ethylene oxide(EtO);this gas reduces the presence of amplifiable DNA and causes minimal interference to downstream HID‑analytical methods.During sample collection,indicating cards,for example,Indicating FTA™(GE Healthcare Life Sciences,UK),are used to collect and store buccal cell DNA.These cards contain a dye which changes color on application of a colorless sample.Generating“DNA‑free”indicating cards using EtO should not impact the dyes’ability to indicate sample location or the efficacy of the card in downstream HID‑analytical methods.This study was initiated to identify alternative dyes to those currently used with sample indicating collection cards.The most promising,dyes when applied to cellulose papers exhibited a uniform color distribution and excellent sample indicating properties even when mixed with chemicals associated with FTA™.When dyed cellulose papers were exposed to EtO,ultraviolet radiation,elevated temperature,and humidity,negligible fading or discoloration was observed.The presence of these dyes on cellulose papers did not interfere with direct short tandem repeat(STR)profiling.Allelic concordance,first pass success rate,and mean peak heights were comparable to samples applied to Indicating FTA.Biological samples applied to EtO‑treated dyed cellulose papers and stored>1 month produced full STR profiles of sufficient quality to allow submission to DNA databases,confirming negligible interference from EtO treatment.These alternative sample indicating dyes resist EtO‑mediated fading while fulfilling the Forensic Community’s expectation for“DNA‑free”with negligible impact on collection card performance.
