登革1型和2型病毒混合感染样本的一步法实时荧光定量PCR检测方法的建立

本文旨在建立检测登革1型和2型病毒的一步法实时荧光定量PCR方法,为登革热的诊断和预测提供技术支撑。依据登革1型和2型病毒的保守区序列设计引物和探针,建立了同时检测两个血清型登革病毒的一步法实时荧光定量PCR方法。特异性实验结果显示,该方法可检测出登1型和2型病毒,与登革3型、4型病毒、黄热病毒、流感H3N2病毒均无交叉反应,具有较好的特异性。对登革1型和登革2型的灵敏度均为102copies/μL。应用该方法,可检测出登革1型和2型病毒混合感染C6/36后不同时间点病毒复制动态。在两个血清型登革病毒混合感染C6/36细胞72~120 h登革2型病毒的拷贝数与单独感染时登革2型病毒的拷贝数相比显著下降。上述研究证实建立的一步法实时荧光定量PCR方法具有特异性好灵敏度高、重复性好等特点,能够用于登革型和2型病毒混合感染样本的检测。     

多重PCR检测方法和液态芯片技术在呼吸道病毒检测中的应用研究

目的 评估澳大利亚维多利亚感染性疾病参比实验室（The Victorian Infectious Diseases Reference Laboratory,VIDRL）的呼吸道病毒多重PCR检测方法和Luminex公司多指标同步分析液态芯片技术-呼吸道病毒检测试剂盒（xTAG RVP）,应用于多种常见呼吸道病毒检测的优势和缺点,为更好地、有针对性地选择合适技术进行临床实验诊断或公共卫生应急检测提供依据.方法 使用VIDRL的呼吸道病毒多重PCR检测方法和Luminex公司xTAG RVP试剂盒对198份临床流感样症状患者的咽拭子标本进行常见呼吸道病毒的核酸检测.结果 198份临床标本经VIDRL多重PCR方法和xTAG RVP方法检测,阳性率分别为38.89％和45.45％,符合率为72.22％.单一病毒的符合率为87.88％～100％.结论 xTAG RVP方法与VIDRL多重PCR方法相比,两者在常见呼吸道病毒检测中的特异性和敏感性相近,xTAG RVP方法具有明显的快速和高通量的优势.     

甲型流感病毒荧光RT-PCR检测方法的设计和检定

目的　设计甲型流感病毒TaqMan荧光RT-PCR检测方法并对其进行检定。方法　用DNAStar和PrimerPremier5. 0软件设计甲型流感病毒荧光PCR检测所用引物和探针 ;在GenBank中进行Blast以及电子对比证明其具有高度的特异性和保守性 ;和标准RT-PCR进行比较 ,检测此方法的灵敏度。结果　所设计的引物和探针高度特异和保守 ,灵敏度比标准RT-PCR方法高 3～ 27倍 ,并且反转录和PCR可合并为一步。结论　设计了甲型流感病毒TaqMan检测方法 ;该方法具有特异、灵敏和简便的特点     

巢式RT-PCR检测甲肝疫苗的感染性滴度研究

目的 建立一种快速灵敏特异的检测甲肝疫苗滴度的巢式RT-PCR检测方法。方法 运用巢式RT-PCR法对甲肝疫苗病毒滴度进行检测，并与细胞培养ELISA检测法进行比较。结果 巢式RT-PCR灵敏度和特异性均与细胞培养ELISA检测法相似。结论 巢式RT-PCR是一种简便、快速、灵敏的甲肝病毒检测方法，用于疫苗的常规检测有较好前景。     

两种新型冠状病毒免疫学检测方法的应用评价

目的：为有效检测新型冠状病毒特异性抗体（新冠抗体），利用化学发光法和胶体金法分别检测已完成中和试验的92份血清，对两种方法的敏感度和特异度等进行初步比较和评价，为大规模人群免疫后血清SARS-CoV-2抗体水平监测提供参考。方法：采用病毒中和试验、胶体金试剂盒和全自动化学发光分析仪分别对样本开展SARS-CoV-2特异性抗体检测，计算诊断特异度、敏感度、阴阳性预测值和符合率。结果：化学发光法的灵敏度为100.00%，高于胶体金法的71.64%；胶体金法的特异度为92.00%，高于化学发光法的64.00%；胶体金法阴阳性预测值分别为54.76%和96.00%，而化学发光法的阴阳性预测值分别为100.00%和88.16%；胶体金法和化学发光法的Kappa值分别为0.525和0.721。结论：以微量病毒中和抗体试验为金标准，化学发光法的灵敏度和Kappa值较高，适用于SARS-CoV-2病例辅助性快速检测；胶体金法测定SARS-CoV-2特异度总抗体的灵敏度一般，特异度较高，其使用简便快速、成本低廉，在条件限制、需要及时监测等情况下，适用于快速大规模筛查以及阳性样本初步检测。随着新冠疫苗在人...     

诺如病毒检测技术研究

诺如病毒是非细菌性腹泻暴发的主要病原之一,对相关检测技术的研究十分必要。本文通过引物、探针、染料的选择,结合适用范围、灵敏度、特异性、重复性的评估对常规反转录-聚合酶链反应、荧光定量RT-PCR、反转录-环介导等温扩增、基因芯片、酶联免疫吸附测定和荧光微球检测条快速检测诺如病毒的技术进行了综述。着重关注了食品和水中诺如病毒检测的病毒富集技术,并提出平衡准确灵敏与简便高效对诺如病毒检测技术的研究具有重要意义。     

Kadipiro病毒TaqmanReal—timePCR检测方法的建立

目的利用TaqManReal—timePCR技术，建立Kadipiro病毒（KDV）实时荧光定量PCR检测方法。方法根据GenBank发表的KDV基因序列资料分析结果，在其第12片段基因区段设计KDV特异引物与探针，利用14株不同科属病毒核酸验证方法特异性，重复实验检验方法稳定性，利用体外转录的定量RNA标准品建立基因拷贝数定量分析模型。结果引物与探针具有良好的特异性，同一样品重复检测D值的变异系数均〈1．8％，定量分析模型的灵敏度为100拷贝／反应。结论建立了一种特异、灵敏、高效的KDV一步法TaqManReal-timePCR检测方法，为今后该病毒的监测与研究工作提供技术手段。     

甲肝病毒特异性TaqMan荧光定量PCR法检测甲肝病毒核酸的研究

目的 建立甲肝病毒(HAY)特异性TaqMan荧光定量PCR检测方法,并对血清等样品中的HAV进行检测.方法 根据参考文献,选取HAV基因组保守区5'-NCR区引物和探针,建立特异性强、敏感度高、稳定性好的TaqMan HAV Real-time RT-PCR检测体系并应用于甲肝患者血清等病毒核酸的检测.结果 本研究建立的方法能够特异检测出样品中的甲肝病毒,其灵敏度介于每反应0.1CCID50至0.01CCID50之间.同一样本重复检测3次,批内样本Ct值的变异系数最大2.0％,批间样本Ct值的变异系数最大2.6％.急性期甲型肝炎患者血清中甲肝病毒为5.18×102～4.93×107拷贝/ml.结论 本实验建立的HAV Real-time RT-PCR检测方法具有特异、灵敏、快速等优点,可成功应用于临床样本等甲肝病毒的病原学检测.     

戊型肝炎病毒TaqMan Real-time RT-PCR法的建立及应用

目的 建立灵敏、特异、稳定的戊型肝炎病毒(HEV) TaqMan Real-time PCR检测方法.方法 根据GenBank中的HEV相关序列,选取HEV基因组ORF2的保守区域设计合成特异性引物和探针,建立TaqMan HEV Real-time RT-PCR检测体系,评价体系的特异性、敏感度和稳定性,并应用于临床样本的检测.结果 本研究建立的HEV Real-time RT-PCR检测体系最低检测极限达到10个拷贝/反应,重复性实验Ct值的变异系数(CV)最大为1.53％,并且该体系能特异检测出戊肝临床样本中的HEV,其拷贝数从1.87×104拷贝/ml到8.12×106拷贝/ml不等.结论 成功建立特异性强、灵敏度高的HEV Real-time RT-PCR检测方法,应用于临床样本检测时取得了良好效果,为HEV分子病原学诊断打下基础.     

诺如病毒快速检测试剂的评价

目的评价德国R—Biopharm公司生产的RIDA QUICK诺如病毒快速检测试剂卡（免疫层析法）的准确性、安全性以及与对照试剂的等效性。方法以上市的IDEA Norovirus检测试剂盒（酶联免疫法）平行测定结果为参考,评价RIDA QUICK诺如病毒快速检测试剂卡（免疫层析法）的灵敏度、特异性及符合率。结果临床检测样本1042份,新型快速检测试剂诺如病毒检测特异性98．4％,灵敏度92．4％,与对照试剂的符合率为97．6％。结论RIDA QUICK诺如病毒快速检测试剂卡对于诺如病毒抗原的检测具有良好的特异性和灵敏度。     

New commercially available PCR and microplate hybridization assay for detection and differentiation of human polyomaviruses JC and BK in cerebrospinal fluid, serum, and urine samples

JC and BK human polyomaviruses (family Polyomaviridae) may cause severe neurological or urinary tract pathologies in immunocompromised hosts. In the present study, we evaluated a new commercially available PCR and microplate colorimetric hybridization assay for the standardized differential detection of JC virus (JCV) and BK virus (BKV) genomes in clinical samples. This JC/BK Consensus test was first evaluated by testing serial dilutions of JCV or BKV plasmid DNA standards and was then compared with an in-house reference PCR assay for the detection of JC and BK virus genomes in 70 cerebrospinal fluid (CSF) samples of patients with neurological disorders and in 75 serum or plasma samples and 125 urine samples of renal graft recipients. This new test allowed a limit of detection of 10 copies and 1 copy of JC and BK virus genomes, respectively, and was able to differentiate various levels of JCV, BKV, and mixed JCV and BKV DNA genomes in a single reaction tube. Our results showed 100% specificity and sensitivity for the JC/BK Consensus test with CSF samples. With serum or plasma samples, this test had a sensitivity and a specificity of 100% for both JCV and mixed JCV and BKV DNA detection and a sensitivity and a specificity of 100 and 97.8% for BKV DNA detection, respectively. With urine samples, the sensitivity and specificity were 100 and 96.6%, respectively, for JCV DNA detection; 100 and 89.4%, respectively, for BKV DNA detection; and 44.4 and 100%, respectively, for mixed JCV and BKV DNA detection. In conclusion, our data indicate that this new test, the JC/BK Consensus test, is valuable for the sensitive and specific differential detection of single JCV and BKV infections in CSF, serum or plasma, and urine samples. The use of this reliable PCR assay would improve the routine virological diagnosis as well as the clinical care of immunocompromised patients with polyomavirus-related pathologies.     

SARS-CoV-2核酸检测试剂盒的制备及样本混检性能分析

目的开发一种适用于中国大规模人群新型冠状病毒(SARS-CoV-2)感染筛查的核酸检测试剂盒,分析对多样本混合检测的效果,为临床多样本混合检测提供理论指导。方法选用SARS-CoV-2的ORF1ab、N 2个靶基因,并加入RNase P作为内标基因进行试剂盒研发。采用单一变量及正交实验优化试剂盒中3个靶基因引物及探针浓度比例。评价试剂盒的准确性、特异性及灵敏度。收集阳性样本与阴性样本按不同比例混合,采用本试剂盒及市售参比试剂盒分别检测混合样本的SARS-CoV-2,比较不同试剂盒检测不同靶标的检出浓度及扩增Ct值。结果本试剂盒阳性符合率、阴性符合率及特异性均为100%,灵敏度为1×10³ copies/mL;不同试剂盒对混合样本中不同靶基因的检出浓度不同,本试剂盒对ORF1ab靶基因的检出限为混合比例1/20,灵敏度高于参比试剂盒(1/10),且检测相同浓度模板的扩增Ct值低于参比试剂盒(P<0.05)。结论本试剂盒ORF1ab靶基因检测灵敏度高,在SARS-CoV-2核酸多样本混合检测中更具优势,适用于大规模人群筛查。     

Direct detection of human cytomegalovirus in urine specimens from renal transplant patients following polymerase chain reaction amplification

A polymerase chain reaction (PCR) assay was used to amplify human cytomegalovirus (HCMV) directly from urine specimens taken from renal transplant patients. In serial urine samples from patients who had at least one specimen positive for HCMV; the PCR assay consistently detected the presence of HCMV DNA sequences, whereas virus detection by other tests such as enzyme-linked immunosorbent assay (ELISA), nonradioactive DNA hybridization assay, and virus isolation were variable. Of 37 specimens positive by PCR, 36 were positive by either ELISA, hybridization assay, or virus isolation. Infectious virus was detected in 13 of the 37 PCR-positive urines. HCMV DNA was detected by PCR in all samples that were positive for HCMV by either hybridization assay or virus isolation. The viral genome copy number was determined by PCR assay for several urine samples that were positive by virus isolation but negative for HCMV by ELISA or hybridization assay. Viral genome copy number estimates indicated the presence of HCMV at very low levels in these urines verifying the fidelity of the virus isolation procedures. The consistency of the PCR assay makes it an ideal method for detection of infection and monitoring antiviral drug therapy in patients infected with HCMV.     

Genotyping of 11 human papillomaviruses by multiplex PCR with a GeXP analyzer

A new, rapid, and high-throughput method was developed for simultaneous detection of 11 human papillomavirus (HPV) genotypes including nine high-risk types (HPV16, 18, 31, 33, 35, 39, 52, 58, and 66) and two low-risk types (HPV6 and 11) in a single tube by multiplex PCR based on a GenomeLab Gene Expression Profiler (GeXP) analyzer (GeXP-PCR). Eleven sets of chimeric primers were used to initiate the PCR, and one pair of universal primers was used for the subsequent cycles of the PCR. The specificity of GeXP-PCR for each HPV type was examined with clinical samples of single type HPV infection tested previously. The sensitivity of GeXP-PCR was evaluated by performing the assay on serial 10-fold dilutions of cloned PCR products. The GeXP-PCR achieved a sensitivity of 100 copies when all of the 11 pre-mixed plasmids containing HPV targets were present. Analyses of 124 clinical specimens using the GeXP-PCR demonstrated that the GeXP-PCR assay had comparable sensitivity and specificity to those of reported multiple PCR assay and an increased detection of HPV 11 in samples with mixed infections. In conclusion, the GeXP-PCR is a fast, sensitive, and high throughput method for the detection of multiple HPV infections.     

Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology

Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.     

登革热病毒RNA实时荧光定量RT-PCR检测方法的建立

目的建立TaqMan探针实时荧光定量RT-PCR方法,测定登革热病毒（DV）及DV病毒的RNA拷贝数。方法利用TaqMan探针,建立实时荧光定量RT-PCR方法,通过对登革热病毒RNA定量外标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量RT-PCR方法的灵敏度、特异性和重复性。结果该方法检测灵敏度可达1×103copies/mL,特异性及重复性良好,对同一样品进行5次重复检测,其循环阈值的平均标准偏差为0.792。结论TaqMan探针实时荧光定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定登革热病毒及DVRNA载量。     

Differential detection of avian oncogenic viruses in poultry layer farms and Turkeys by use of multiplex PCR

Avian oncogenic viruses include Marek's disease virus (MDV), a highly contagious herpesvirus, as well as retroviruses such as avian leukosis virus (ALV) subgroups A to J and reticuloendotheliosis virus (REV). In this study, we examined the incidence of these viruses in suspected samples collected from poultry layer farms of South India, mainly in the Namakkal district of Tamil Nadu, a highly dense poultry-growing area in India. The histopathology-positive tissue sections were identified and further confirmed by immunohistochemistry using virus-specific antibodies. The viruses belonging to all 3 groups (MDV, ALV, and REV) were isolated in a cell culture system and confirmed by immunofluorescence using virus-specific antibodies. PCR appeared to be the method of choice for rapid and accurate diagnosis of these viruses. The multiplex PCR primers specific to MDV, ALV, REV, and chicken DNA were designed for rapid differential diagnosis. The specificity of the primers was checked by amplification of DNA from virus-infected cell culture in comparison with uninfected samples, and sensitivity was evaluated by calculating the minimum copy number at which amplification occurs in the cloned PCR products. The sequences of the amplicons were compared by BLAST analysis. PCR tests demonstrated the presence of single, dual, or triple viruses in some of the samples. Of 169 samples screened by multiplex PCR, 9 samples were positive for MDV, 17 samples were positive for ALV, 12 samples were positive for REV, and 17 samples were positive for both ALV and REV. Three samples were positive for all three viruses. ALV-positive samples were further subjected to subgroup-specific PCR, which gave positive results for subgroups B and D but not for subgroup J. Multiplex PCR appeared to be a useful technique for rapid differential diagnosis of avian oncogenic viruses and detection of multiple infections of avian oncogenic viruses under field conditions.     

Rapid multiplex nested PCR for detection of respiratory viruses

Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken.