Objective: To determine the diagnostic performancesof six Chinese PCR kits for detection of Chlamydiatrachomatis in patients with sexually transmitteddiseases using cell culture and LCR as references.Methods: Endocerv...Objective: To determine the diagnostic performancesof six Chinese PCR kits for detection of Chlamydiatrachomatis in patients with sexually transmitteddiseases using cell culture and LCR as references.Methods: Endocervical or urethral swab specimenswere collected from 673 patients attending STDclinics in Beijing, Shanghai, Nanjing and Tianjin. C.trachomatis culture and PCR were performed onspecimens from all patients while LCR detection wasperformed only on specimens with discordant cultureand PCR results.Results: Of the 616 patients, 6.3% (39) wereculture-positive while 23.5% to 28.7% were positiveby PCR testing. Compared to cell culture, the sensi-tivity of all six PCR methods was 90% or higher. In200 cases with discrepant reports, LCR and PCRshowed excellent consistency (YI index: 0.523-0.881 ), the sensitivity and specificity of PCR methodswere 83.9%- 98.6% and 66.7%- 94.7% respectively,while PCR2 showed the highest YI index (0.881). Withthe reference standard defined as culture positive orLCR positive plus at least one PCR positive fordiscrepant results, we found that the specificity andsensitivity of all six Chinese PCR kits were higherthan 95% and 85%, respectively.Conclusions: Domestically-produced PCR kits forChlamydia trachomatis detection are highly sensi-tive and specific, however, quality control remainsimportant in their clinical application.展开更多
目的认定一个人类白细胞抗原(HLA)新等位基因。方法应用多聚酶链反应-序列特异性寡核苷酸技术(polymerase chain reaction-sequence specific oligonucleotide,PCR-SSO)基因分型发现可能的HLA新等位基因,用PCR直接测序及针对组特异性扩...目的认定一个人类白细胞抗原(HLA)新等位基因。方法应用多聚酶链反应-序列特异性寡核苷酸技术(polymerase chain reaction-sequence specific oligonucleotide,PCR-SSO)基因分型发现可能的HLA新等位基因,用PCR直接测序及针对组特异性扩增产物测序,确认与最同源HLA等位基因序列的差异。结果发现一个样本的HLA-A位点结果异常,其核苷酸序列与已知所有HLA-A位点等位基因序列不一致,与同源性最高的等位基因A*030103差异是在第2外显子区域的256位碱基发生了C→G的替换,257位碱基发生了A→G的替换,270位碱基发生了T→A的替换,导致相应的62位密码子由CAG→GGG,编码的氨基酸由谷氨酰胺(Gln)→甘氨酸(Gly),导致相应的66位密码子由AAT→AAA,编码的氨基酸由天冬氨酸(Asn)→赖氨酸(Lys)。结论该等位基因为HLA-A位点的一个新等位基因,2006年7月13日被WHOHLA因子命名委员会正式命名为HLA-A*0323。展开更多
In the present study,the partial gene sequences of P32 protein,an immunogenic envelope protein of Capripoxviruses (CaPV),were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates,and r...In the present study,the partial gene sequences of P32 protein,an immunogenic envelope protein of Capripoxviruses (CaPV),were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates,and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains.A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene.The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels.Phylogenetic analysis showed three distinct clusters of SPPV,GTPV and Lumpy skin disease virus (LSDV) isolates.Further,multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I,which is present in SPPV isolates studied.This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains.The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV.The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods.展开更多
[Objective]This study aimed to establish a multiplex PCR detection method of herbicide-tolerant canola.[Method] An endogenous reference gene(CruA) and three exogenous genes(T-CaMV 35 S, P-CaMV 35 S and pat) were selec...[Objective]This study aimed to establish a multiplex PCR detection method of herbicide-tolerant canola.[Method] An endogenous reference gene(CruA) and three exogenous genes(T-CaMV 35 S, P-CaMV 35 S and pat) were selected for multiplex PCR. Specific primers were designed based on national standards or related literature. The annealing temperature, ratio of primer concentration and sensitivity of the established multiplex PCR system were optimized. The optimal multiplex PCR system was verified with known samples. [Result] The experimental results showed that the optimal annealing temperature of multiplex PCR was 58 ℃; the optimal ratio of primer concentration(μmol/L) was T-CaMV 35S: CruA: P-CaMV 35S: pat=0.1: 0.2: 0.2: 0.2;the detection sensitivity of the established multiplex PCR method was 0.3 ng. The amplified bands of known samples were completely consistent with the molecular characteristics. [Conclusion] This study provided a rapid, accurate and effective multiplex PCR technique for detection of herbicide-tolerant canola.展开更多
文摘Objective: To determine the diagnostic performancesof six Chinese PCR kits for detection of Chlamydiatrachomatis in patients with sexually transmitteddiseases using cell culture and LCR as references.Methods: Endocervical or urethral swab specimenswere collected from 673 patients attending STDclinics in Beijing, Shanghai, Nanjing and Tianjin. C.trachomatis culture and PCR were performed onspecimens from all patients while LCR detection wasperformed only on specimens with discordant cultureand PCR results.Results: Of the 616 patients, 6.3% (39) wereculture-positive while 23.5% to 28.7% were positiveby PCR testing. Compared to cell culture, the sensi-tivity of all six PCR methods was 90% or higher. In200 cases with discrepant reports, LCR and PCRshowed excellent consistency (YI index: 0.523-0.881 ), the sensitivity and specificity of PCR methodswere 83.9%- 98.6% and 66.7%- 94.7% respectively,while PCR2 showed the highest YI index (0.881). Withthe reference standard defined as culture positive orLCR positive plus at least one PCR positive fordiscrepant results, we found that the specificity andsensitivity of all six Chinese PCR kits were higherthan 95% and 85%, respectively.Conclusions: Domestically-produced PCR kits forChlamydia trachomatis detection are highly sensi-tive and specific, however, quality control remainsimportant in their clinical application.
文摘目的认定一个人类白细胞抗原(HLA)新等位基因。方法应用多聚酶链反应-序列特异性寡核苷酸技术(polymerase chain reaction-sequence specific oligonucleotide,PCR-SSO)基因分型发现可能的HLA新等位基因,用PCR直接测序及针对组特异性扩增产物测序,确认与最同源HLA等位基因序列的差异。结果发现一个样本的HLA-A位点结果异常,其核苷酸序列与已知所有HLA-A位点等位基因序列不一致,与同源性最高的等位基因A*030103差异是在第2外显子区域的256位碱基发生了C→G的替换,257位碱基发生了A→G的替换,270位碱基发生了T→A的替换,导致相应的62位密码子由CAG→GGG,编码的氨基酸由谷氨酰胺(Gln)→甘氨酸(Gly),导致相应的66位密码子由AAT→AAA,编码的氨基酸由天冬氨酸(Asn)→赖氨酸(Lys)。结论该等位基因为HLA-A位点的一个新等位基因,2006年7月13日被WHOHLA因子命名委员会正式命名为HLA-A*0323。
文摘In the present study,the partial gene sequences of P32 protein,an immunogenic envelope protein of Capripoxviruses (CaPV),were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates,and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains.A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene.The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels.Phylogenetic analysis showed three distinct clusters of SPPV,GTPV and Lumpy skin disease virus (LSDV) isolates.Further,multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I,which is present in SPPV isolates studied.This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains.The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV.The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods.
基金Supported by Special Fund for Modern Agricultural Technology Innovation and Demonstration of Sichuan Province(2014CXSF-040)General Natural Science Project of the Education Department of Sichuan Province(15ZB0331)
文摘[Objective]This study aimed to establish a multiplex PCR detection method of herbicide-tolerant canola.[Method] An endogenous reference gene(CruA) and three exogenous genes(T-CaMV 35 S, P-CaMV 35 S and pat) were selected for multiplex PCR. Specific primers were designed based on national standards or related literature. The annealing temperature, ratio of primer concentration and sensitivity of the established multiplex PCR system were optimized. The optimal multiplex PCR system was verified with known samples. [Result] The experimental results showed that the optimal annealing temperature of multiplex PCR was 58 ℃; the optimal ratio of primer concentration(μmol/L) was T-CaMV 35S: CruA: P-CaMV 35S: pat=0.1: 0.2: 0.2: 0.2;the detection sensitivity of the established multiplex PCR method was 0.3 ng. The amplified bands of known samples were completely consistent with the molecular characteristics. [Conclusion] This study provided a rapid, accurate and effective multiplex PCR technique for detection of herbicide-tolerant canola.
