人博卡病毒和人偏肺病毒核酸检测方法的建立及初步应用
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摘要
儿童急性呼吸道感染(ARTI)是世界范围内小儿最常见的致病和致死原因之一,在过去几十年里,由于更先进仪器的应用及高灵敏性检测方法的建立,使得越来越多的呼吸道病毒被人们发现,人博卡病毒和人偏肺病毒即是近些年新发现的呼吸道病原,从而进一步丰富了呼吸道感染的病原普。本研究的主要内容及研究结果具体如下:
1.根据人博卡病毒(Human Bocavirus,HBoV) NP-1基因和人偏肺病毒(Human Metapneumovirus,hMPV) M基因的保守序列设计用于常规PCR/RT-PCR的引物,扩增HBoV NP-1基因和hMPV M基因片段,建立HBoV和hMPV的常规PCR/RT-PCR检测方法,然后对反应条件进行优化并对检测方法的灵敏性和特异性进行评价。本研究所建立的常规PCR/RT-PCR检测方法检测HBoV NP-1基因和hMPV的M基因其灵敏度分别为250 DNA copies/reaction和500 DNA copies/reaction,且结果具有良好的特异性。
2.根据HBoV和hMPV基因的保守序列设计用于实时荧光定量PCR/RT-PCR的引物和TaqMan探针。建立HBoV和hMPV的实时荧光定量PCR/RT-PCR(real-time Fluorescent Quantitative PCR/RT-PCR)检测方法并对建立的方法进行敏感性、特异性、重复稳定性实验。本研究所建立的实时荧光定量PCR方法检测HBoV灵敏度为10 DNA copies/reaction;实时荧光定量RT-PCR方法检测hMPV灵敏度可达4.95 DNA copies/reaction,线性范围可达109-100 copies/reaction。且检测结果都具有良好的稳定性和可重复性,对临床其他常见呼吸道病毒检测未出现特异性扩增曲线,说明该检测方法的特异性良好。
3.用所建立的方法对由沈阳儿童医院采集来的76份临床标本中的HBoV和hMPV进行平行检测,将两种检测方法的结果进行比较分析并探索其临床意义。用实时荧光定量PCR方法对76例临床标本进行检测,共检出HBoV阳性5例,高于常规PCR检测结果3例,且常规PCR检出结果包含在荧光定量PCR检出结果中;常规RT-PCR与实时荧光定量RT-PCR方法对76份临床样本进行平行检测,检出hMPV阳性样本分别为(5/76)和(7/76)且常规RT-PCR检出结果包含在荧光定量RT-PCR检出结果中。说明两种检测方法有很好的符合率,相对于常规PCR/RT-PCR,荧光定量PCR/RT-PCR检测方法灵敏性、特异性更好。
结论
实验结果表明,本研究初步建立了针对儿童急性呼吸道感染的HBoV和hMPV的常规PCR/RT-PCR检测方法和实时荧光定量PCR/RT-PCR检测方法,并初步证明了所建立的方法具有灵敏、特异、快速的特点,将两种检测方法用于临床样本平行检测,结果显示两种检测方法具有较好的一致性,但实时荧光定量PCR/RT-PCR检测方法操作更简便、自动化程度更高、检测灵敏性更好、检测速度更快,可替代常规PCR/RT-PCR检测方法,具有在临床上应用的可行性及较好的应用前景。
Among children,acute respiratory tract infections(ARTIs)are a major cause of morbidity and mortality worldwide.In the past few decades, due to the more advanced instruments application and more sensitive detection methods, more respiratory viruses were found such as Human Bocavirus and Human Metapneumovirus.However,there are also some respiratory tract illnesses in childern that the pathogens can not be identified from respiratory samples.The major content and reasults of our research as follows:
1. To establish two conventional PCR/RT-PCR assays separately for Human Bocavirus(HBoV) and Human Metapneumovirus(hMPV),and to detect HBoV and hMPV from nasopharyngeal samples of children with ARTI. The sensitivity of the established conventional PCR/RT-PCR for HBoV NP1 gene and hMPV M gene were 250 DNA copies/reaction and 500 DNA copies/reaction respectively with a better specificity.
2. The specific primers and Taqman probes used for real-time Fluorescent Quantitative PCR/RT-PCR were designed respectively according to the conservative sequence.Subsequently, experiments were undertaken to assess diagnostic criteria such as specificity, sensitivity and reproducibility. The sensitivity of real-time Fluorescent Quantitative PCR assay for HBoV was 10 DNA copies/reaction and the sensitivity of real-time Fluorescent Quantitative RT-PCR assay for hMPV was 4.95 DNA copies/reaction,both of the realts with the better specificity and reproducibility.
3. A total of 76 nasopharyngeal swab specimens derived from Shenyang were screened for the presence of HBoV and hMPV using conventional PCR/RT-PCR and real-time Fluorescent Quantitative PCR/RT-PCR respectively.
5 specimens were identified positive for HBoV by real-time Fluorescent Quantitative PCR assay,and 3 positive specimens screened by conventional PCR,and the specimens identified by conventional PCR were contained in the specimens identified by real-time Fluorescent Quantitative PCR assay.As for the hMPV,7 positive specimens were detected by real-time Fluorescent Quantitative RT-PCR with a higher dectection rate than that identified by conventional RT-PCR assay(5/76),and the result of the conventional PCR were according with that of real-time Fluorescent Quantitative RT-PCR assay.
Conclusions
The nucleic acid assays for detection of HBoV and hMPV have been developed and were preliminary proved to have a better sensitivity、specificity、reproducibility and these assays maybe applied for epidemical surveillance and clinical diagnosis of HBoV and HMPV.The comparison results of convential PCR/RT-PCR and realtime Fluorescent Quantitative PCR/RT-PCR showed that realtime Fluorescent Quantitative PCR/RT-PCR is easy to operate,faster and more sensitive than convertial PCR/RT-PCR.
1.根据人博卡病毒(Human Bocavirus,HBoV) NP-1基因和人偏肺病毒(Human Metapneumovirus,hMPV) M基因的保守序列设计用于常规PCR/RT-PCR的引物,扩增HBoV NP-1基因和hMPV M基因片段,建立HBoV和hMPV的常规PCR/RT-PCR检测方法,然后对反应条件进行优化并对检测方法的灵敏性和特异性进行评价。本研究所建立的常规PCR/RT-PCR检测方法检测HBoV NP-1基因和hMPV的M基因其灵敏度分别为250 DNA copies/reaction和500 DNA copies/reaction,且结果具有良好的特异性。
2.根据HBoV和hMPV基因的保守序列设计用于实时荧光定量PCR/RT-PCR的引物和TaqMan探针。建立HBoV和hMPV的实时荧光定量PCR/RT-PCR(real-time Fluorescent Quantitative PCR/RT-PCR)检测方法并对建立的方法进行敏感性、特异性、重复稳定性实验。本研究所建立的实时荧光定量PCR方法检测HBoV灵敏度为10 DNA copies/reaction;实时荧光定量RT-PCR方法检测hMPV灵敏度可达4.95 DNA copies/reaction,线性范围可达109-100 copies/reaction。且检测结果都具有良好的稳定性和可重复性,对临床其他常见呼吸道病毒检测未出现特异性扩增曲线,说明该检测方法的特异性良好。
3.用所建立的方法对由沈阳儿童医院采集来的76份临床标本中的HBoV和hMPV进行平行检测,将两种检测方法的结果进行比较分析并探索其临床意义。用实时荧光定量PCR方法对76例临床标本进行检测,共检出HBoV阳性5例,高于常规PCR检测结果3例,且常规PCR检出结果包含在荧光定量PCR检出结果中;常规RT-PCR与实时荧光定量RT-PCR方法对76份临床样本进行平行检测,检出hMPV阳性样本分别为(5/76)和(7/76)且常规RT-PCR检出结果包含在荧光定量RT-PCR检出结果中。说明两种检测方法有很好的符合率,相对于常规PCR/RT-PCR,荧光定量PCR/RT-PCR检测方法灵敏性、特异性更好。
结论
实验结果表明,本研究初步建立了针对儿童急性呼吸道感染的HBoV和hMPV的常规PCR/RT-PCR检测方法和实时荧光定量PCR/RT-PCR检测方法,并初步证明了所建立的方法具有灵敏、特异、快速的特点,将两种检测方法用于临床样本平行检测,结果显示两种检测方法具有较好的一致性,但实时荧光定量PCR/RT-PCR检测方法操作更简便、自动化程度更高、检测灵敏性更好、检测速度更快,可替代常规PCR/RT-PCR检测方法,具有在临床上应用的可行性及较好的应用前景。
Among children,acute respiratory tract infections(ARTIs)are a major cause of morbidity and mortality worldwide.In the past few decades, due to the more advanced instruments application and more sensitive detection methods, more respiratory viruses were found such as Human Bocavirus and Human Metapneumovirus.However,there are also some respiratory tract illnesses in childern that the pathogens can not be identified from respiratory samples.The major content and reasults of our research as follows:
1. To establish two conventional PCR/RT-PCR assays separately for Human Bocavirus(HBoV) and Human Metapneumovirus(hMPV),and to detect HBoV and hMPV from nasopharyngeal samples of children with ARTI. The sensitivity of the established conventional PCR/RT-PCR for HBoV NP1 gene and hMPV M gene were 250 DNA copies/reaction and 500 DNA copies/reaction respectively with a better specificity.
2. The specific primers and Taqman probes used for real-time Fluorescent Quantitative PCR/RT-PCR were designed respectively according to the conservative sequence.Subsequently, experiments were undertaken to assess diagnostic criteria such as specificity, sensitivity and reproducibility. The sensitivity of real-time Fluorescent Quantitative PCR assay for HBoV was 10 DNA copies/reaction and the sensitivity of real-time Fluorescent Quantitative RT-PCR assay for hMPV was 4.95 DNA copies/reaction,both of the realts with the better specificity and reproducibility.
3. A total of 76 nasopharyngeal swab specimens derived from Shenyang were screened for the presence of HBoV and hMPV using conventional PCR/RT-PCR and real-time Fluorescent Quantitative PCR/RT-PCR respectively.
5 specimens were identified positive for HBoV by real-time Fluorescent Quantitative PCR assay,and 3 positive specimens screened by conventional PCR,and the specimens identified by conventional PCR were contained in the specimens identified by real-time Fluorescent Quantitative PCR assay.As for the hMPV,7 positive specimens were detected by real-time Fluorescent Quantitative RT-PCR with a higher dectection rate than that identified by conventional RT-PCR assay(5/76),and the result of the conventional PCR were according with that of real-time Fluorescent Quantitative RT-PCR assay.
Conclusions
The nucleic acid assays for detection of HBoV and hMPV have been developed and were preliminary proved to have a better sensitivity、specificity、reproducibility and these assays maybe applied for epidemical surveillance and clinical diagnosis of HBoV and HMPV.The comparison results of convential PCR/RT-PCR and realtime Fluorescent Quantitative PCR/RT-PCR showed that realtime Fluorescent Quantitative PCR/RT-PCR is easy to operate,faster and more sensitive than convertial PCR/RT-PCR.
引文
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