禽流感和新城疫病毒二重荧光定量RT-PCR检测方法的建立

目的：建立二重荧光定量RT-PCR方法,用于禽流感病毒（AIV）和新城疫病毒（NDV）的检测。方法：根据AIV和NDV的基因保守序列,设计了AIV和NDV的2对特异性引物和2条用不同荧光基团标记的TaqMan探针;对反应条件和试剂浓度进行优化,建立了能够同时检测AIV和NDV的Z-重荧光定量RT-PCR方法。结果：所建方法特异性好,对AIV和NDV的检测敏感性均达到2000个模板拷贝数,比常规RT-PCR敏感性高100倍;抗干扰能力强,对AIV和NDV不同模板浓度进行组合,仍可有效地同时检测2种病毒。对保存的AIV或NDV鸡胚尿囊液及临床病料进行二重荧光定量RT-PCR检测,结果尿囊液检测的拷贝数均达到10^10/μL以上,临床病料的拷贝数为2．13x10^8-6．52x10^4／μL。结论：建立了用于检测AIV和NDV的二重荧光定量RT-PCR法,该方法特异、敏感、快速、可定量,对AIV和NDV的防制有重要意义。     

多重RT-PCR用于临床检测三种胃肠炎病毒的研究

轮状病毒、诺瓦克病毒和星状病毒是引起病毒性胃肠炎的主要病原因子。研究采用JV12/JV13、P1/P2和Mon340/Mon348三对引物,建立了同时检测这3种病毒的多重RT-PCR技术,并应用于128份临床粪便样本的检测,检出轮状病毒62份(48.44%),诺瓦克病毒8份(6.25%),星状病毒11份(8.59%)。在灵敏度试验中,轮状病毒的检测灵敏度为5pg/mL、诺瓦克病毒和星状病毒的检测灵敏度均为50pg/mL。该研究所建立3种常见胃肠炎病毒的多重RT-PCR方法具有特异性强、灵敏度高的特点,可用于临床病原诊断和溯源。     

细胞培养结合实时荧光RT-PCR法快速检测甲肝病毒滴度的研究

目的建立一种细胞培养与实时荧光RT-PCR相结合的快速检测甲肝病毒滴度的方法。方法根据甲肝病毒(HAV)L-A-1株5'端基因组序列,设计了2条基因特异性引物及一条探针,建立实时荧光RT-PCR法,结合细胞培养检测甲肝病毒滴度,并与ELISA检测法进行比较。结果实验中建立的方法能特异检测甲肝病毒,细胞培养8d检测病毒滴度为lg107.0CCID50/mL。同一样本重复检测3次,批内样本Ct值的变异系数最大为0.89%,批间样本Ct值变异系数最大为1.66%。建立的细胞培养结合实时荧光RT-PCR法(细胞培养8 d)与细胞培养ELISA法(细胞培养28 d)检测甲肝病毒滴度结果差异无统计学意义(P0.05)。结论该方法具有快速、灵敏、特异等优点,应用于疫苗常规检测有良好前景。     

Performance of Simplexa Dengue Molecular Assay Compared to Conventional and SYBR Green RT-PCR for Detection of Dengue Infection in Indonesia

Diagnostic tests based on detection of dengue virus (DENV) genome are available with varying sensitivities and specificities. The Simplexa Dengue assay (Focus Diagnostics) is a newly developed real-time RT-PCR method designed to detect and serotype DENV simultaneously. To assess the performance of the Simplexa Dengue assay, we performed comparison with conventional RT-PCR and SYBR Green real-time RT-PCR on patients sera isolated from eight cities across Indonesia, a dengue endemic country. A total of 184 sera that were confirmed using NS1 and/or IgM and IgG ELISA were examined. Using conventional and SYBR Green real-time RT-PCR, we detected DENV in 53 (28.8%) and 81 (44.0%) out of 184 sera, respectively. When the Simplexa Dengue assay was employed, the detection rate was increased to 76.6% (141 out of 184 samples). When tested in 40 sera that were confirmed by virus isolation as the gold standard, the conventional RT-PCR yielded 95% sensitivity while the sensitivity of SYBR Green real-time RT-PCR and Simplexa Dengue assay reached 97.5% and 100%, respectively. The specificities of all methods were 100% when tested in 43 non-dengue illness and 20 healthy human samples. Altogether, our data showed the higher detection rate of Simplexa Dengue compared to conventional and SYBR Green real-time RT-PCR in field/surveillance setting. In conclusion, Simplexa Dengue offers rapid and accurate detection and typing of dengue infection and is suitable for both routine diagnostic and surveillance.     

The Development and Application of the Two Real-Time RT-PCR Assays to Detect the Pathogen of HFMD

Large-scale Hand, Foot, and Mouth Disease (HFMD) outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs). Among them, human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1–2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID₅₀/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells). The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11) by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.     

ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples

This study applied the integrated cell culture/polymerase chain reaction methodology (ICC/PCR) for rapid and specific detection of both cytopathogenic and noncytopathogenic viruses. Results of this study showed that the use of direct RT-PCR or conventional cell culture alone may yield erroneous results with the analysis of environmental samples. The purpose of this study was to compare cultural, molecular, and combined assays for the most effective method of virus detection in variable environmental samples. Using ICC/PCR, stock enterovirus inocula of > or =10 PFU were PCR positive in at least 4/5 replicate flasks after only 5 h of incubation in cell culture, and in all flasks after > or =10 h. An inoculum of one PFU was detected by PCR after 20 h of cell culture incubation while for concentrations of virus below one PFU, 25 h of incubation was sufficient. Similarly, hepatitis A virus (HAV) inocula of 100 MPN/flask, produced indeterminate CPE in cell culture, but were clearly detected by ICC/PCR following 48 h of incubation. Lower levels of HAV, 1 and 10 MPN, were detected by ICC/PCR after 96 to 72 h of incubation, respectively. Cell culture lysates from 11 environmental sample concentrates of sewage, marine water, and surface drinking water sources, were positive for enteroviruses by ICC/PCR compared to 3 positive by direct RT-PCR alone. Results from ICC/PCR eventually agreed with cell culture but required < or =48 h of incubation, compared to as long as 3 weeks for CPE following incubation with BGM and FRhK cells.     

GeXP多重PCR技术同时检测12种常见呼吸道病毒

本研究建立了一种基于GeXP多重基因表达遗传分析系统的多重RT-PCR检测方法,该方法可以同时检测12种呼吸道病毒,包括流感病毒A型和B型、季节性H1N1、副流感病毒1～3型、人鼻病毒、人偏肺病毒、腺病毒、呼吸道合胞病毒A型和B型、人博卡病毒。针对病原体保守区序列设计12种病毒的特异性引物,分别用已验证的阳性标本为模板检验多重体系的特异性。多重检测体系在10~3拷贝/μL水平可同时检测到12种病毒。另检测24份临床标本,以real-time RT-PCR为参考标准,进一步验证检测体系。结果表明,这种基于GeXP系统的新方法灵敏度高、特异性强,可以快速同时检测12种常见呼吸道病毒。     

Validation of a real-time RT-PCR method to quantify Newcastle Disease Virus (NDV) titer and comparison with other quantifiable methods

A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 (TCID50) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.     

Rapid detection and monitoring therapeutic efficacy of Mycobacterium tuberculosis complex using a novel real-time assay

We combined real-time RT-PCR and real-time PCR (R/P) assays using a hydrolysis probe to detect Mycobacterium tuberculosis complex (MTBC)-specific 16S rRNA and its rRNA gene (rDNA). The assay was applied to 28 nonrespiratory and 207 respiratory specimens from 218 patients. Total nucleic acids (including RNA and DNA) were extracted from samples, and results were considered positive if the repeat RT-PCR threshold cycle was < or =35 and the ratio of real-time RT-PCR and real-time PCR load was > or =1.51. The results were compared with those from existing methods, including smear, culture, and real-time PCR. Following resolution of the discrepant results between R/P assay and culture, the overall sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) of all samples (including nonrespiratory and respiratory specimens) were 98.2%, 97.2%, 91.7%, and 99.4%, respectively, for R/P assay, and 83.9%, 89.9%, 72.3%, and 94.7%, respectively, for real-time PCR. Furthermore, the R/P assay of four patient samples showed a higher ratio before treatment than after several days of treatment. We conclude that the R/P assay is a rapid and accurate method for direct detection of MTBC, which can distinguish viable and nonviable MTBC, and thus may guide patient therapy and public health decisions.