Raw poultry and poultry products are a significant source of zoonotic bacterial pathogen transmission;thus the sensitive detection of major zoonotic pathogens (Salmonella spp., Campylobacter jejuni, and Listeria monoc...Raw poultry and poultry products are a significant source of zoonotic bacterial pathogen transmission;thus the sensitive detection of major zoonotic pathogens (Salmonella spp., Campylobacter jejuni, and Listeria monocytogenes) is a vital food safety issue. Recently, third generation PCR technology, known as droplet digital PCR (ddPCR) has been developed to be more accurate and sensitive to detect genetic targets than current quantification methods, but this technology has not been tested within an industrial setting. There is an on-going study within our laboratory is investigating the effects of sampling times and sampling methods on the cultural and molecular (via qPCR) quantification of dominant zoonotic pathogens within a poultry processing facility. This presents a unique opportunity to compare the quantification resulted from this emerging, third generation technology to traditional quantification methods currently employed by the poultry industry. The results show that ddPCR detected pathogen-specific genes from more pathogen:sampling time combinations than either the qPCR or culturing methods from the final scalder and chiller tanks at three stages of processing (Start, Mid, and End). In fact, both ddPCR and qPCR substantially outperformed culture methods commonly used in poultry processing food safety-related studies, with Salmonella recovered only from the Mid and End sampling times from the scalder tank. While neither C. jejuni nor L. monocytogenes were recovered culturally, ddPCR was able to detect their respective genes commonly throughout the processing day in both the scalder and chiller water samples. Additionally, the use of unfiltered processing water provided significantly greater detection of bacterial and pathogen-specific gene abundances than did an analysis of larger volumes of filtered water. Considering the ddPCR-derived concentrations of the bacterial pathogens were consistent with what was previously found culturally in commercial poultry processing operations, ddPCR represented a signi展开更多
本研究利用微滴式数字PCR(droplet digital polymerase chain reaction,dd PCR)技术对食品和饲料中鹅源性成分进行检测与精确定量。选取鹅的单拷贝核基因作为靶基因,建立了鹅源性成分靶基因拷贝数与样品质量之间的线性关系;选取21种常...本研究利用微滴式数字PCR(droplet digital polymerase chain reaction,dd PCR)技术对食品和饲料中鹅源性成分进行检测与精确定量。选取鹅的单拷贝核基因作为靶基因,建立了鹅源性成分靶基因拷贝数与样品质量之间的线性关系;选取21种常见动物物种及鹅的9个品系对所设计的引物及探针的种间特异性和种内保守性进行了验证,并对该方法的检测下限(limit of detection,LOD)和定量检测下限(limit of quantification,LOQ)进行了验证,利用已知鹅源性成分含量的样品和市售商品对该方法的准确性和适用性分别进行了验证。结果表明,所设计的引物及探针具有良好的种间特异性和种内保守性,该定量检测方法的检测下限和定量检测下限分别为1 copy/μL和5 copies/μL,且该方法具有良好的准确性和适用性,可用于对食品和饲料中鹅源性成分进行检测和精确定量。展开更多
以大肠菌群的lacZ基因为靶基因,建立一种快速、稳定、灵敏、特异的微滴数字聚合酶链式反应(Droplet Digital Polymerase Chain Reaction,ddPCR)定量检测方法。对ddPCR反应体系中试剂浓度、退火温度等条件优化筛选的同时,考察了方法的线...以大肠菌群的lacZ基因为靶基因,建立一种快速、稳定、灵敏、特异的微滴数字聚合酶链式反应(Droplet Digital Polymerase Chain Reaction,ddPCR)定量检测方法。对ddPCR反应体系中试剂浓度、退火温度等条件优化筛选的同时,考察了方法的线性范围、精密度和定量限。最终确定反应体系中的最佳引物和探针浓度分别为0.2和0.5μmol/L,最佳退火温度为56℃。大肠菌群lacZ基因组DNA浓度范围为3.95～7.80×10~4拷贝(copies)/20μL ddPCR反应液时,方法的线性关系良好(R^2=0.999)。文章所建立的方法可检出每微升单个拷贝数的大肠菌群lacZ基因组DNA,且具有快速测定、灵敏度高、特异性强、重复性良好等特点,为建立水污染早期应急机制提供了重要的参考价值。展开更多
目的构建一种重组新型冠状病毒疫苗(腺病毒载体)病毒颗粒数微滴式数字PCR(droplet digital PCR,ddPCR)检测方法。方法以新型冠状病毒BA.1株基因组为靶基因,选取刺突蛋白保守序列设计引物和探针,建立重组新型冠状病毒疫苗病毒颗粒数ddPC...目的构建一种重组新型冠状病毒疫苗(腺病毒载体)病毒颗粒数微滴式数字PCR(droplet digital PCR,ddPCR)检测方法。方法以新型冠状病毒BA.1株基因组为靶基因,选取刺突蛋白保守序列设计引物和探针,建立重组新型冠状病毒疫苗病毒颗粒数ddPCR方法,并进行线性、准确度、专属性、重复性和耐用性等方法学验证。结果ddPCR的最佳退火温度是63℃,病毒颗粒数为5×10^(5)~2×10^(7) VP/ml时,线性和回收率良好,探针和引物特异性好,重复性和中间精密度变异系数(coefficient of variation,CV)都在10%以内,耐用性CV值在15%以内,并且与qPCR结果相比,CV值都在10%以内。结论本研究建立的ddPCR方法灵敏度高、稳定性好、特异性强,可用于重组新型冠状病毒疫苗(腺病毒载体)病毒颗粒数的定量检测。展开更多
目的 基于分泌卷曲相关蛋白2(secreted frizzled-related protein 2,SFRP2)基因,评价荧光定量法(Methy Light)、微滴数字PCR(droplet digital PCR,ddPCR)、核酸质谱、靶向亚硫酸氢盐二代测序4种甲基化检测方法。方法 对4种甲基化检测方...目的 基于分泌卷曲相关蛋白2(secreted frizzled-related protein 2,SFRP2)基因,评价荧光定量法(Methy Light)、微滴数字PCR(droplet digital PCR,ddPCR)、核酸质谱、靶向亚硫酸氢盐二代测序4种甲基化检测方法。方法 对4种甲基化检测方法的检测限(limit of detection, LOD)、定量限(limit of quantitation, LOQ)及稳定性方面进行比较,其中稳定性用变异系数(coefficient of variation,CV)来评估。结果 SFRP2基因在Methy Light、ddPCR、核酸质谱和靶向亚硫酸氢盐二代测序中的LOD分别为1.2500ng/孔、0.0625ng/孔、0.0625ng/孔、1.2500ng/孔,在LOD方面,ddPCR和核酸质谱的表现较好;SFRP2基因在MethyLight、ddPCR、核酸质谱和靶向亚硫酸氢盐二代测序中的LOQ分别为0.800%、0.032%、4.000%、0.032%,在LOQ方面,ddPCR和靶向亚硫酸氢盐二代测序的表现较好;SFRP2基因在MethyLight、ddPCR、核酸质谱和靶向亚硫酸氢盐二代测序中的变异系数分别为2.72%、0.68%、0.73%、0.15%,在稳定性方面,靶向亚硫酸氢盐二代测序的表现最好。结论 ddPCR的甲基化检测在检测限、定量限稳定性和经济性方面具有优越性,能够稳定地检测出肿瘤细胞的痕量游离DNA,在液态活检中具有广泛应用前景。展开更多
文摘Raw poultry and poultry products are a significant source of zoonotic bacterial pathogen transmission;thus the sensitive detection of major zoonotic pathogens (Salmonella spp., Campylobacter jejuni, and Listeria monocytogenes) is a vital food safety issue. Recently, third generation PCR technology, known as droplet digital PCR (ddPCR) has been developed to be more accurate and sensitive to detect genetic targets than current quantification methods, but this technology has not been tested within an industrial setting. There is an on-going study within our laboratory is investigating the effects of sampling times and sampling methods on the cultural and molecular (via qPCR) quantification of dominant zoonotic pathogens within a poultry processing facility. This presents a unique opportunity to compare the quantification resulted from this emerging, third generation technology to traditional quantification methods currently employed by the poultry industry. The results show that ddPCR detected pathogen-specific genes from more pathogen:sampling time combinations than either the qPCR or culturing methods from the final scalder and chiller tanks at three stages of processing (Start, Mid, and End). In fact, both ddPCR and qPCR substantially outperformed culture methods commonly used in poultry processing food safety-related studies, with Salmonella recovered only from the Mid and End sampling times from the scalder tank. While neither C. jejuni nor L. monocytogenes were recovered culturally, ddPCR was able to detect their respective genes commonly throughout the processing day in both the scalder and chiller water samples. Additionally, the use of unfiltered processing water provided significantly greater detection of bacterial and pathogen-specific gene abundances than did an analysis of larger volumes of filtered water. Considering the ddPCR-derived concentrations of the bacterial pathogens were consistent with what was previously found culturally in commercial poultry processing operations, ddPCR represented a signi
文摘本研究利用微滴式数字PCR(droplet digital polymerase chain reaction,dd PCR)技术对食品和饲料中鹅源性成分进行检测与精确定量。选取鹅的单拷贝核基因作为靶基因,建立了鹅源性成分靶基因拷贝数与样品质量之间的线性关系;选取21种常见动物物种及鹅的9个品系对所设计的引物及探针的种间特异性和种内保守性进行了验证,并对该方法的检测下限(limit of detection,LOD)和定量检测下限(limit of quantification,LOQ)进行了验证,利用已知鹅源性成分含量的样品和市售商品对该方法的准确性和适用性分别进行了验证。结果表明,所设计的引物及探针具有良好的种间特异性和种内保守性,该定量检测方法的检测下限和定量检测下限分别为1 copy/μL和5 copies/μL,且该方法具有良好的准确性和适用性,可用于对食品和饲料中鹅源性成分进行检测和精确定量。
文摘目的构建一种重组新型冠状病毒疫苗(腺病毒载体)病毒颗粒数微滴式数字PCR(droplet digital PCR,ddPCR)检测方法。方法以新型冠状病毒BA.1株基因组为靶基因,选取刺突蛋白保守序列设计引物和探针,建立重组新型冠状病毒疫苗病毒颗粒数ddPCR方法,并进行线性、准确度、专属性、重复性和耐用性等方法学验证。结果ddPCR的最佳退火温度是63℃,病毒颗粒数为5×10^(5)~2×10^(7) VP/ml时,线性和回收率良好,探针和引物特异性好,重复性和中间精密度变异系数(coefficient of variation,CV)都在10%以内,耐用性CV值在15%以内,并且与qPCR结果相比,CV值都在10%以内。结论本研究建立的ddPCR方法灵敏度高、稳定性好、特异性强,可用于重组新型冠状病毒疫苗(腺病毒载体)病毒颗粒数的定量检测。
文摘目的 基于分泌卷曲相关蛋白2(secreted frizzled-related protein 2,SFRP2)基因,评价荧光定量法(Methy Light)、微滴数字PCR(droplet digital PCR,ddPCR)、核酸质谱、靶向亚硫酸氢盐二代测序4种甲基化检测方法。方法 对4种甲基化检测方法的检测限(limit of detection, LOD)、定量限(limit of quantitation, LOQ)及稳定性方面进行比较,其中稳定性用变异系数(coefficient of variation,CV)来评估。结果 SFRP2基因在Methy Light、ddPCR、核酸质谱和靶向亚硫酸氢盐二代测序中的LOD分别为1.2500ng/孔、0.0625ng/孔、0.0625ng/孔、1.2500ng/孔,在LOD方面,ddPCR和核酸质谱的表现较好;SFRP2基因在MethyLight、ddPCR、核酸质谱和靶向亚硫酸氢盐二代测序中的LOQ分别为0.800%、0.032%、4.000%、0.032%,在LOQ方面,ddPCR和靶向亚硫酸氢盐二代测序的表现较好;SFRP2基因在MethyLight、ddPCR、核酸质谱和靶向亚硫酸氢盐二代测序中的变异系数分别为2.72%、0.68%、0.73%、0.15%,在稳定性方面,靶向亚硫酸氢盐二代测序的表现最好。结论 ddPCR的甲基化检测在检测限、定量限稳定性和经济性方面具有优越性,能够稳定地检测出肿瘤细胞的痕量游离DNA,在液态活检中具有广泛应用前景。
