Porcine epidemic diarrhea,a highly contagious enteric infectious disease caused by the porcine epidemic diarrhea virus（ PEDV） with symptoms of vomit,diarrhea,loss of appetite of suckling pig,has led to serious econo...Porcine epidemic diarrhea,a highly contagious enteric infectious disease caused by the porcine epidemic diarrhea virus（ PEDV） with symptoms of vomit,diarrhea,loss of appetite of suckling pig,has led to serious economic loss to the global swine industry. In this study,a real-time fluorescence reverse transcription loop-mediated isothermal amplification（ RT-LAMP） assay was developed to detect PEDV RNA. The real-time fluorescence RT-LAMP assay was performed at62 ℃ for 60 min,using a simple and portable device,the ESE-Quant Tube Scanner. The detection limit of RNA was 2. 9 × 10~6 copies/μl,10 times as sensitive as RT-PCR,and the detection was specific only to PEDV. Application of this method to clinical samples yielded a positivity rate of 93%,which was higher than that of RT-PCR. This technique saves time and is efficient,and is thus expected to be useful for the diagnosis of PEDV infection in the field.展开更多
为了建立高效、灵敏的猪流行性腹泻病毒(PEDV)检测方法,本研究从GenBank数据库中获取PEDV N基因序列,扩增出PEDV N基因标准质粒,并在N基因的保守区域内设计了一对特异性荧光定量引物,成功建立了SYBR Green I实时荧光定量PCR检测方法。...为了建立高效、灵敏的猪流行性腹泻病毒(PEDV)检测方法,本研究从GenBank数据库中获取PEDV N基因序列,扩增出PEDV N基因标准质粒,并在N基因的保守区域内设计了一对特异性荧光定量引物,成功建立了SYBR Green I实时荧光定量PCR检测方法。经过一系列试验表明,该检测方法线性关系良好,R^(2)值为0.99;特异性强,敏感性高,最低可检测至2.23 copies/μL,比普通PCR灵敏约100倍;重复性好,组内变异系数为0.25%~0.43%,组间变异系数为0.67%~0.97%;对于各地区96份临床样品检测出PEDV阳性率为25%。本研究建立的实时荧光定量PCR检测方法为PEDV的临床诊断、流行病学调查以及定量研究提供了有效的检测工具。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
基金Supported by Science and Technology Research Project of Universities in Hebei Province,China(QN2014220)
文摘Porcine epidemic diarrhea,a highly contagious enteric infectious disease caused by the porcine epidemic diarrhea virus（ PEDV） with symptoms of vomit,diarrhea,loss of appetite of suckling pig,has led to serious economic loss to the global swine industry. In this study,a real-time fluorescence reverse transcription loop-mediated isothermal amplification（ RT-LAMP） assay was developed to detect PEDV RNA. The real-time fluorescence RT-LAMP assay was performed at62 ℃ for 60 min,using a simple and portable device,the ESE-Quant Tube Scanner. The detection limit of RNA was 2. 9 × 10~6 copies/μl,10 times as sensitive as RT-PCR,and the detection was specific only to PEDV. Application of this method to clinical samples yielded a positivity rate of 93%,which was higher than that of RT-PCR. This technique saves time and is efficient,and is thus expected to be useful for the diagnosis of PEDV infection in the field.
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.
