[ Objective] To improve the accuracy and efficiency of the detection which used in Perkinsus sp and Marteilia refringens, and then short- en the detective cycle. [ Method] According to the gene sequence of Perkinsus s...[ Objective] To improve the accuracy and efficiency of the detection which used in Perkinsus sp and Marteilia refringens, and then short- en the detective cycle. [ Method] According to the gene sequence of Perkinsus sp and Marteilia refringens from gene bank, design two pairs of spe- cific primers and two TaqMan probes with different fluorophores labeled. Optimizing the reactive conditions and reagent concentration in order that establishing the duplex real-time PCR method for detecting Perkinsus sp and Marteilia refringens simultaneously. [ Result ] The sensitivity of the du- plex real-time PCR method which about Pertdnsus sp and Marteilia refringens is 40 template copies. After combine the templates of Perkinsus sp and Marteilia refringens with different concentrations, this method still could be detect this two protozoan efficiently and synchronously. [ Condudon] The es- tablished duplex real-time PCR method for detecting Perkinsus sp. and Marteilia refringens possesses lots of advantages, such as specific, sensitive, rapid, quantitative and reproducible, can be used for clinical detection of infection which was caused by Perkinsus sp. and Marteilia refringens.展开更多
Porcine circoviruses(PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-i...Porcine circoviruses(PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-infections in clinical samples. Thus, it would be practical to develop a duplex PCR method to detect PCV2 and PCV3 simultaneously. In this study, specific primers and probes were designed to target PCV2 cap and PCV3 rep genes. A duplex real-time PCR method was then developed to detect the two viruses. The assay was found to be highly specific, sensitive, and reproducible for PCV2/3 without cross-reactions with other swine pathogens. The sensitivity of this assay was 2.9 copies for the PCV2 plasmid and 22.5 copies for the PCV3 plasmid. The established assay was then used to detect PCV2/3 infection in 340 clinical samples collected in the first half of 2017. The results showed that the co-infection rate of PCV2/3 in the samples was 27.6%. Our study provides an important tool that can be used to perform urgently needed surveys for the two porcine circoviruses to evaluate their impact on the swine industry.展开更多
Objective: To develop a duplex real-time PCR assay for pharmaceutical rapid microbial detection of Staphylococcus aureus and Pseudomonas aeruginosa. Methods: The specific primers and probes were designed to amplify th...Objective: To develop a duplex real-time PCR assay for pharmaceutical rapid microbial detection of Staphylococcus aureus and Pseudomonas aeruginosa. Methods: The specific primers and probes were designed to amplify the femB gene of S. aureus and the DNA gyrase subunit B gene of P. aeruginosa. The sensitivity of the system was detected by a multiple proportional dilution method. In order to examine the specificity of the system, other twenty-one bacteria strains were assayed simultaneously. Results: A highly sensitive and specific duplex real-time PCR assay for the detection of S. aureus and P. aeruginosa was established. The sensitivity was 50 copies/μL. The specificity was 100%. The whole detection procedure can be finished within 2.5 h. Conclusion: The duplex real-time PCR method is efficient in detecting with good sensitivity and specificity. There is a good prospect of this method applying in disease prevention and pharmaceutical industry due to the simultaneous detection of two pathogens.展开更多
Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(I...Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(ICV),and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology.To date,the prevalence of ICV and IDV in China is unclear,but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential.To efficiently monitor ICV and IDV,it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research.A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed.TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed.This method exhibited good specificity and sensitivity,and the detection limit reached 1 × 10^(1) copies/pL of plasmid standards of each pathogen.Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method.One positive sample of IDV was detected,and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing.The duplex real-time PCR detection method represents a sensitive and specific tool to detect IG/and IDV,It provides technical support for virus research and clinical diagnosis of ICV and IDV.This information will benefit animal and human health.展开更多
应用SYBR Green I染料能选择性结合双链DNA的特点,可检测到沙门氏菌fimI基因特异性靶序列扩增所产生的荧光信号,通过熔解曲线可知其熔点值约为85.6℃,而对其他非沙门氏菌则检测不到荧光信号。建立了一种肉品中的沙门氏菌Real-time PCR...应用SYBR Green I染料能选择性结合双链DNA的特点,可检测到沙门氏菌fimI基因特异性靶序列扩增所产生的荧光信号,通过熔解曲线可知其熔点值约为85.6℃,而对其他非沙门氏菌则检测不到荧光信号。建立了一种肉品中的沙门氏菌Real-time PCR检测方法,用该方法检测市售牛肉、香肠中的沙门氏菌,其检测灵敏度分别为13,12 cfu/25 g,从样品的处理到得出检验结果可以在10 h内完成。该检测方法具有简便、快速、特异性强、敏感度高等特点。展开更多
目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-...目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-Ti me PCR方法有很好的特异性与敏感性,所检测沙门菌结果均为阳性,而非沙门菌均为阴性;标准曲线相关系数为R2=0.993,其敏感性为5CFU。运用该方法对108份鸡粪便、50份鸡肉以及58份水样进行检测,阳性率分别为3.7%(6/108)、4%(2/50)和3.4%(2/58),与传统细菌分离检测结果相符。结论结果表明该方法具有简便、快速、特异性强、敏感性高等特点,此研究为环境及疾病诊断中沙门菌快速检测提供了新方法。展开更多
为定量检测猪流行性腹泻病毒(PEDV)载量,建立PEDV的Real-time PCR方法。用RT-PCR方法扩增PEDV的M基因片段,构建含有M基因片段的重组质粒,用该重组质粒进行SYBR Green I Real-time PCR,来建立检测PEDV的荧光定量PCR方法。结果显示:在(5.5...为定量检测猪流行性腹泻病毒(PEDV)载量,建立PEDV的Real-time PCR方法。用RT-PCR方法扩增PEDV的M基因片段,构建含有M基因片段的重组质粒,用该重组质粒进行SYBR Green I Real-time PCR,来建立检测PEDV的荧光定量PCR方法。结果显示:在(5.56×102～5.56×107)拷贝/μL范围内,所建立方法具有优良的线性关系,其决定系数为0.9996,扩增效率为99.5%,扩增产物的熔解曲线只有一个特异性峰,无引物二聚体峰,熔解温度为(81.18±0.21)℃。该方法对猪传染性胃肠炎病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、伪狂犬病病毒、猪圆环病毒II型等猪源病毒均检测不到扩增产物,重复性试验的变异系数小于3%,对临床样品的检出率高于常规PCR方法。研究结果表明:建立的Real-time PCR检测方法特异性强、重复性好、灵敏性高,可用于PEDV的定量检测及其早期感染的快速诊断。展开更多
文摘[ Objective] To improve the accuracy and efficiency of the detection which used in Perkinsus sp and Marteilia refringens, and then short- en the detective cycle. [ Method] According to the gene sequence of Perkinsus sp and Marteilia refringens from gene bank, design two pairs of spe- cific primers and two TaqMan probes with different fluorophores labeled. Optimizing the reactive conditions and reagent concentration in order that establishing the duplex real-time PCR method for detecting Perkinsus sp and Marteilia refringens simultaneously. [ Result ] The sensitivity of the du- plex real-time PCR method which about Pertdnsus sp and Marteilia refringens is 40 template copies. After combine the templates of Perkinsus sp and Marteilia refringens with different concentrations, this method still could be detect this two protozoan efficiently and synchronously. [ Condudon] The es- tablished duplex real-time PCR method for detecting Perkinsus sp. and Marteilia refringens possesses lots of advantages, such as specific, sensitive, rapid, quantitative and reproducible, can be used for clinical detection of infection which was caused by Perkinsus sp. and Marteilia refringens.
基金supported by Grants from the National Key Research and Development Program (2016YFD0500703)Major Science and Technology Projects in Henan Province (171100110200)Luoyang HeLuo Talent Plan (Dr. Kegong Tian)
文摘Porcine circoviruses(PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-infections in clinical samples. Thus, it would be practical to develop a duplex PCR method to detect PCV2 and PCV3 simultaneously. In this study, specific primers and probes were designed to target PCV2 cap and PCV3 rep genes. A duplex real-time PCR method was then developed to detect the two viruses. The assay was found to be highly specific, sensitive, and reproducible for PCV2/3 without cross-reactions with other swine pathogens. The sensitivity of this assay was 2.9 copies for the PCV2 plasmid and 22.5 copies for the PCV3 plasmid. The established assay was then used to detect PCV2/3 infection in 340 clinical samples collected in the first half of 2017. The results showed that the co-infection rate of PCV2/3 in the samples was 27.6%. Our study provides an important tool that can be used to perform urgently needed surveys for the two porcine circoviruses to evaluate their impact on the swine industry.
文摘Objective: To develop a duplex real-time PCR assay for pharmaceutical rapid microbial detection of Staphylococcus aureus and Pseudomonas aeruginosa. Methods: The specific primers and probes were designed to amplify the femB gene of S. aureus and the DNA gyrase subunit B gene of P. aeruginosa. The sensitivity of the system was detected by a multiple proportional dilution method. In order to examine the specificity of the system, other twenty-one bacteria strains were assayed simultaneously. Results: A highly sensitive and specific duplex real-time PCR assay for the detection of S. aureus and P. aeruginosa was established. The sensitivity was 50 copies/μL. The specificity was 100%. The whole detection procedure can be finished within 2.5 h. Conclusion: The duplex real-time PCR method is efficient in detecting with good sensitivity and specificity. There is a good prospect of this method applying in disease prevention and pharmaceutical industry due to the simultaneous detection of two pathogens.
基金This work was financially supported by the National Key Research and Development Program of China(2017YFD0500101)the Fundamental Research Funds for the Central Universities(Y0201900459).
文摘Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(ICV),and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology.To date,the prevalence of ICV and IDV in China is unclear,but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential.To efficiently monitor ICV and IDV,it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research.A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed.TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed.This method exhibited good specificity and sensitivity,and the detection limit reached 1 × 10^(1) copies/pL of plasmid standards of each pathogen.Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method.One positive sample of IDV was detected,and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing.The duplex real-time PCR detection method represents a sensitive and specific tool to detect IG/and IDV,It provides technical support for virus research and clinical diagnosis of ICV and IDV.This information will benefit animal and human health.
文摘目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-Ti me PCR方法有很好的特异性与敏感性,所检测沙门菌结果均为阳性,而非沙门菌均为阴性;标准曲线相关系数为R2=0.993,其敏感性为5CFU。运用该方法对108份鸡粪便、50份鸡肉以及58份水样进行检测,阳性率分别为3.7%(6/108)、4%(2/50)和3.4%(2/58),与传统细菌分离检测结果相符。结论结果表明该方法具有简便、快速、特异性强、敏感性高等特点,此研究为环境及疾病诊断中沙门菌快速检测提供了新方法。
文摘为定量检测猪流行性腹泻病毒(PEDV)载量,建立PEDV的Real-time PCR方法。用RT-PCR方法扩增PEDV的M基因片段,构建含有M基因片段的重组质粒,用该重组质粒进行SYBR Green I Real-time PCR,来建立检测PEDV的荧光定量PCR方法。结果显示:在(5.56×102～5.56×107)拷贝/μL范围内,所建立方法具有优良的线性关系,其决定系数为0.9996,扩增效率为99.5%,扩增产物的熔解曲线只有一个特异性峰,无引物二聚体峰,熔解温度为(81.18±0.21)℃。该方法对猪传染性胃肠炎病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、伪狂犬病病毒、猪圆环病毒II型等猪源病毒均检测不到扩增产物,重复性试验的变异系数小于3%,对临床样品的检出率高于常规PCR方法。研究结果表明:建立的Real-time PCR检测方法特异性强、重复性好、灵敏性高,可用于PEDV的定量检测及其早期感染的快速诊断。
