目的:建立Taq M an-MGB探针Real-tim e荧光PCR快速检测单增李斯特菌技术。方法:在对单增李斯特菌invA序列进行分析、比较基础上,设计一对特异性Taq M an-MGB探针法引物及探针,通过Real-tim ePCR反应条件和反应体系的优化,实现对单增李...目的:建立Taq M an-MGB探针Real-tim e荧光PCR快速检测单增李斯特菌技术。方法:在对单增李斯特菌invA序列进行分析、比较基础上,设计一对特异性Taq M an-MGB探针法引物及探针,通过Real-tim ePCR反应条件和反应体系的优化,实现对单增李斯特菌的快速检测;用克隆到pMD18-T载体上的李斯特菌invA基因阳参片段及不同菌株、食品标本验证方法的特异性和敏感性。结果:方法的灵敏性高,其循环阈值与模板浓度的对数值具有很好的对应关系,最低可检测57个拷贝数,经18 h增菌,可检测低至4.5 cfu/m l的细菌;特异性强,检测6株单增李斯特菌标准株和100株单增李斯特菌样品分离株的PCR循环域值(CT值)均小于25,而90株威氏李斯特菌、英诺克李斯特菌、绵羊李斯特菌以及非李斯特菌PCR循环域值(CT值)大于35;快速,最快1 h可出结果,实际样品20 h可出结果,而常规细菌培养法需要一周以上;对103份速冻米面食品进行检测,13份阳性,与常规方法无显著性差异(P>0.05)。结论:Taq M an-MGB探针的单增李斯特菌Real-tim e荧光PCR检测方法具有特异性强,敏感性高,易操作等优点,可推广应用。展开更多
A real-time RT-PCR assay using Taq Man-MGB probes was developed to detect and type the bovine viral diarrhea virus（BVDV） in cattle.Universal primers and Taq Man-MGB probes were designed from the 5′-untranslated reg...A real-time RT-PCR assay using Taq Man-MGB probes was developed to detect and type the bovine viral diarrhea virus（BVDV） in cattle.Universal primers and Taq Man-MGB probes were designed from the 5′-untranslated region of known pestiviral sequences.Prior to optimizing the assay, c RNAs were transcribed in vitro from the BVDV 1 and BVDV 2 RTPCR products to make standard curves.The detection limit of the assay was 1.72×102 copies for BVDV 1 and 2.14×102copies for BVDV 2.The specificity of the assay evaluated on several BVDV strains including bovine herpesvirus 1（BHV 1）, foot and mouth disease virus（FMDV） and several classical swine fever virus（CSFV） strains showed specific detection of the positive virus over 40 cycles.The assay was highly reproducible with the coefficient of variance ranging from 1.04 to 1.33% for BVDV 1 and from 0.83 to 1.48% for BVDV 2, respectively.Using this method, we tested a total of 2 327 cattle from three dairy farms for the presence of BVDV persistently infected（PI） animals.In this assay, each RT-PCR template contained a mixture of ten samples from different animals.The occurrence rate of PI cattle in three farms ranging from 0.9 to 2.54% could represent partly the PI rates in cattle farm in China.In conclusion, using our real-time PCR assay, we could effectively detect and type BVDV and identify PI cattle in a rapid and cost-effective manner.展开更多
Infectious bursal disease(IBD)is an important contagious viral infection of immune system of poultry.This infection possesses a permanent threat to the profitability of poultry industry worldwide.The aim of this work ...Infectious bursal disease(IBD)is an important contagious viral infection of immune system of poultry.This infection possesses a permanent threat to the profitability of poultry industry worldwide.The aim of this work was to modify the Taq Man-MGB real-time reverse transcription-polymerase chain reaction(rRT-PCR)in one step involving two fluorogenic Taq Man labeled probe and using this protocol for detection of infectious bursal disease virus(IBDV)collected from suspected cases distributed in different regions of the country during the period 2013-2016.The intralaboratory validation of modified method was realized for specificity,linearity,repeatability,sensitivity and reproducibility.It allowed reducing the test running time by six folds.This method was applied on 102 pools of bursa of fabricius(BF)samples collected from affected broiler farms suspected to be infected by IBDV.Birds showing macroscopic lesions including muscle petechial hemorrhages,hypertrophy and hemorrhage of BF,were subjected to molecular analysis using modified protocol“Taq Man-MGB rRT-PCR”.The validation satisfied all criteria and the assay developed could be a useful tool for a very rapid diagnosis of IBDV and permit to detect and to discriminate in one-step very virulent(vv)from non-vv(classic and variant)IBDV strains.Out of 84 IBDV positive samples,a prevalence of 39%for vv strains and 61%for classical strains was noted.These results indicate that despite the vaccination against IBDV,the vv form of this pathologie continues to cause serious problems for Moroccan broiler chickens.The obtained results indicate the successfully detection of IBDV and differentiated all vvIBDV strains from non-vvIBDV strains;Avian infectious agent RNA viruses tested are negative,demonstrating great specificity of the assay.The results obtained indicate that this method is suitable as a routine laboratory test for the rapid detection and differentiation of IBDV strains in samples of avian origin.展开更多
文摘目的:建立Taq M an-MGB探针Real-tim e荧光PCR快速检测单增李斯特菌技术。方法:在对单增李斯特菌invA序列进行分析、比较基础上,设计一对特异性Taq M an-MGB探针法引物及探针,通过Real-tim ePCR反应条件和反应体系的优化,实现对单增李斯特菌的快速检测;用克隆到pMD18-T载体上的李斯特菌invA基因阳参片段及不同菌株、食品标本验证方法的特异性和敏感性。结果:方法的灵敏性高,其循环阈值与模板浓度的对数值具有很好的对应关系,最低可检测57个拷贝数,经18 h增菌,可检测低至4.5 cfu/m l的细菌;特异性强,检测6株单增李斯特菌标准株和100株单增李斯特菌样品分离株的PCR循环域值(CT值)均小于25,而90株威氏李斯特菌、英诺克李斯特菌、绵羊李斯特菌以及非李斯特菌PCR循环域值(CT值)大于35;快速,最快1 h可出结果,实际样品20 h可出结果,而常规细菌培养法需要一周以上;对103份速冻米面食品进行检测,13份阳性,与常规方法无显著性差异(P>0.05)。结论:Taq M an-MGB探针的单增李斯特菌Real-tim e荧光PCR检测方法具有特异性强,敏感性高,易操作等优点,可推广应用。
基金supported by the China Agriculture Research System(CARS-37)
文摘A real-time RT-PCR assay using Taq Man-MGB probes was developed to detect and type the bovine viral diarrhea virus（BVDV） in cattle.Universal primers and Taq Man-MGB probes were designed from the 5′-untranslated region of known pestiviral sequences.Prior to optimizing the assay, c RNAs were transcribed in vitro from the BVDV 1 and BVDV 2 RTPCR products to make standard curves.The detection limit of the assay was 1.72×102 copies for BVDV 1 and 2.14×102copies for BVDV 2.The specificity of the assay evaluated on several BVDV strains including bovine herpesvirus 1（BHV 1）, foot and mouth disease virus（FMDV） and several classical swine fever virus（CSFV） strains showed specific detection of the positive virus over 40 cycles.The assay was highly reproducible with the coefficient of variance ranging from 1.04 to 1.33% for BVDV 1 and from 0.83 to 1.48% for BVDV 2, respectively.Using this method, we tested a total of 2 327 cattle from three dairy farms for the presence of BVDV persistently infected（PI） animals.In this assay, each RT-PCR template contained a mixture of ten samples from different animals.The occurrence rate of PI cattle in three farms ranging from 0.9 to 2.54% could represent partly the PI rates in cattle farm in China.In conclusion, using our real-time PCR assay, we could effectively detect and type BVDV and identify PI cattle in a rapid and cost-effective manner.
文摘Infectious bursal disease(IBD)is an important contagious viral infection of immune system of poultry.This infection possesses a permanent threat to the profitability of poultry industry worldwide.The aim of this work was to modify the Taq Man-MGB real-time reverse transcription-polymerase chain reaction(rRT-PCR)in one step involving two fluorogenic Taq Man labeled probe and using this protocol for detection of infectious bursal disease virus(IBDV)collected from suspected cases distributed in different regions of the country during the period 2013-2016.The intralaboratory validation of modified method was realized for specificity,linearity,repeatability,sensitivity and reproducibility.It allowed reducing the test running time by six folds.This method was applied on 102 pools of bursa of fabricius(BF)samples collected from affected broiler farms suspected to be infected by IBDV.Birds showing macroscopic lesions including muscle petechial hemorrhages,hypertrophy and hemorrhage of BF,were subjected to molecular analysis using modified protocol“Taq Man-MGB rRT-PCR”.The validation satisfied all criteria and the assay developed could be a useful tool for a very rapid diagnosis of IBDV and permit to detect and to discriminate in one-step very virulent(vv)from non-vv(classic and variant)IBDV strains.Out of 84 IBDV positive samples,a prevalence of 39%for vv strains and 61%for classical strains was noted.These results indicate that despite the vaccination against IBDV,the vv form of this pathologie continues to cause serious problems for Moroccan broiler chickens.The obtained results indicate the successfully detection of IBDV and differentiated all vvIBDV strains from non-vvIBDV strains;Avian infectious agent RNA viruses tested are negative,demonstrating great specificity of the assay.The results obtained indicate that this method is suitable as a routine laboratory test for the rapid detection and differentiation of IBDV strains in samples of avian origin.
