SYBR GreenⅠ实时荧光定量RT-PCR测定肠道病毒71型（EV71）RNA拷贝数方法的建立

目的建立SYBR GreenⅠ荧光染料实时定量RT-PCR方法,测定实验动物等来源的EV71病毒RNA。方法运用EV71VP1保守区引物,优化real time RT-PCR条件,运用NASBA方法扩增EV71病毒RNA,计算拷贝数,经10倍系列稀释做出标准曲线,作为EV71病毒RNA定量检测的外标准品。结果应用Qiagen公司QuantiTect SYBR Green RT-PCR Kit,该标准品可精确定量到100copies/μL,PCR扩增效率达到99.5%。结论 SYBRGreenⅠ荧光染料实时定量PCR法测定EV71病毒RNA拷贝数的方法敏感性高、稳定性好,可用于EV71病毒RNA载量的定量测定。     

Detection of human enteroviruses with real-time PCR assay using TaqMan fluorescent probe

Infections with human enteroviruses are common worldwide and cause a wide range of signs and symptoms. Nowadays in current diagnostics procedures older virological methods, such virus isolation in a cell cultures and seroneutralisation assay, are replaced with molecular biology tests. The aim of the study was development of real-time PCR assay for detection of human adenoviruses. DNA isolated from MK2 cell line infected with nineteen different enterovirus strains was used for development of a qualitative real-time PCR assay using primers targeting a conserved region of the 5'UTR region and a specific TaqMan probe. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of Coxackie A9 cDNA in range between 10 degrees and 10(-8). For comparison typical end-point detected RT-PCR for enterovirus detection with the same cDNA dilutions was made. The sensitivity of novel method was about ten thousand-fold higher than older one. The conclusion is that real-time PCR is very advisable in diagnostics of diseases caused with enteroviruses. The high level of sensitivity, specificity, accuracy, and rapidity provided by this assay are favorable for the use in the detection of enteroviral RNA in clinical specimens, especially from neuroinfections.     

Evaluation of a Viral Microarray Based on Simultaneous Extraction and Amplification of Viral Nucleotide Acid for Detecting Human Herpesviruses and Enteroviruses

In this study, a viral microarray based assay was developed to detect the human herpesviruses and enteroviruses associated with central nervous system infections, including herpes simplex virus type 1, type 2 (HSV1 and HSV2), Epstein-Barr virus (EBV), cytomegalovirus (CMV), enterovirus 71 (EV71), coxsackievirus A 16 (CA16) and B 5(CB5). The DNA polymerase gene of human herpesviruses and 5’-untranslated region of enteroviruses were selected as the targets to design primers and probes. Human herpesviruses DNA and enteroviruses RNA were extracted simultaneously by using a guanidinium thiocyanate acid buffer, and were subsequently amplified through a biotinylated asymmetry multiplex RT-PCR with the specific primer of enteroviruses. In total, 90 blood samples and 49 cerebrospinal fluids samples with suspected systemic or neurological virus infections were investigated. Out of 139 samples, 66 were identified as positive. The specificities of this multiplex RT-PCR microarray assay were over 96% but the sensitivities were various from 100% for HSV1, HSV2, EV71 and CB5, 95.83% for CMV, 80% for EBV to 71.43% for CA16 in comparison with reference standards of TaqMan qPCR/qRT-PCR. The high Kappa values (>0.90) from HSV1, HSV2, CMV, EV71 and CB5 were obtained, indicating almost perfect agreement in term of the 5 viruses detection. But lower Kappa values for EBV (0.63) and CA16 (0.74) displayed a moderate to substantial agreement. This study provides an innovation of simultaneous extraction, amplification, hybridization and detection of DNA viruses and RNA viruses with simplicity and specificity, and demonstrates a potential clinical utility for a variety of viruses’ detection.     

Validation of real-time RT-PCR assays for mRNA quantification in baboons

Real-time RT-PCR has been used widely, both in fundamental research and in clinical diagnostics, for instance for quantification of RNA levels in human tissues and tissue biopsies. In the present study we provide a strategy to validate primers/probes for real-time RT-PCR quantification of baboon samples. The method is based on the TaqMan system and uses primers/probes that have been designed and validated for human real-time RT-PCR. A prerequisite for the accuracy of this strategy is a similar amplification efficiency between human and baboon PCR reactions. We propose two different methods, i.e. by calculating PCR efficiencies from the slope of a dilution curve or by using the linear regression method, to compare the amplification efficiency between human and baboon samples. In conclusion, by performing a simple validation experiment, real-time PCR assays based on human sequences, which are easily available, can be applied for analysis of baboon samples.     

Development of an enterovirus specific PCR method for the quantification of enterovirus genomes in blood of diabetes patients

Background: Insulin-dependent diabetes mellitus or type 1 diabetes is a disease with a diverse aetiology. Epidemiological studies examining newly diagnosed, recent onset IDDM patients have suggested a role for viruses in the aetiology of IDDM (Yoon, 1995, Diabetes/Metabolism Reviews 11, 83–107). Important candidates are the enteroviruses, in particular coxsackieviruses B3 and B4. The latter can cause diabetes in animals (Clements et al., 1995, Lancet 346, 221–223).Objectives: We have developed a quantitative PCR method for the detection of enterovirus genomes in biological samples. The quantitative PCR will be used to screen for enteroviruses in blood of diabetes patients and their relatives by testing a Blood Diabetes Register.Study design: A substantial amount of data has been collected on enterovirus induced IDDM, our study is original in so far as it will be: (1) a quantitative study, not only the presence of viral genome sequences in blood will be determined, but also their concentrations (viral load); and (2) a longitudinal study, samples are and will be collected as a function of time. Positive PCR samples will be quantified using the standard addition method.Results: The test is specific for enteroviruses, since all enteroviruses were detected with equal sensitivity. Viruses belonging to other picornavirus genera scored negative (even up to 3×10⁶ genome copies). An equal detection limit of 10 genome copies was found for all enteroviruses.Conclusions: The developed method will permit us to generate quantitative and longitudinal data of enterovirus genomes in blood of diabetes patients and their relatives, which might help in the elucidation of the relationship between enteroviruses and IDDM.     

Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units

Quantification of viral infectious units is traditionally measured by methods based on forming plaques in semisolid media (PFU) or endpoint dilution of a virus-containing solution (TCID₅₀), methods that are laborious, time-consuming and take on average 3–7 days to carry out. Quantitative real-time PCR is an established method to quantify nucleic acids at high accuracy and reproducibility, routinely used for virus detection and identification. In the present study, a procedure was developed using a two-step real-time PCR and the SYBR Green detection method to study whether there are correlations between TCID₅₀/ml, PFU/ml and Ct values generated by real-time PCR enabling rapid and efficient calculation of titer equivalents when working with viruses in the research laboratory. In addition, an external standard with known concentrations was included using in vitro transcribed viral RNA, thus allowing the calculation of the amount of RNA copies needed for various applications (i.e. per plaque or TCID₅₀).The results show that there is a correlation between the three quantification methods covering a wide range of concentration of viruses. Furthermore, a general regression line between TCID₅₀ and Ct values was obtained for all viruses included in the study, which enabled recording titer equivalents using real-time PCR. Finally, by including an external standard, the amount of RNA genomes generating one TCID₅₀ or PFU for each enterovirus serotype included was determined.     

Assessment of enterovirus contamination in mussel samples from Morocco

Summary In Morocco, shellfish sanitary quality analysis does not currently include enteric virus detection. Enteroviruses are classically detected by cell culture, but molecular methods such as RT-PCR are now broadly used alone or associated to cell culture. RT-PCR has the advantage of requiring less time and budget than cell culture. Bivalve mussels, being filter-feeders tend to accumulate viruses in contaminated seawater. In order to assess mussel contamination by enteroviruses, we screened samples of two origins, an aquaculture system and an area where wild mussels grow. Domestic sewage samples from an outlet near the above wild mussels growing area were also analysed. Viruses were concentrated from mussel meat by direct glycine elution and PEG 8000 precipitation. Total RNA was then extracted from the PEG precipitate by the guanidine thiocyanate method and used in RT-PCR. Enterovirus genomes were detected in 10% of wild shellfish samples, whilst none was present in the aquaculture samples. Since organisms harvested in both growing areas are used for human consumption, the enterovirus contamination observed in this study may highlight a potential public health risk and illustrate the importance of including routine virological analysis of shellfish in Morocco.     

含非竞争性内标四重荧光RT-PCR方法检测手足口肠道病毒方法的研究

旨在了解手足口病的流行和感染情况,并进行快速准确的检测。建立了含非竞争性内标的同时检测肠道病毒通用型、肠道病毒EV71型及柯萨奇病毒CA16型的四重荧光RT-PCR方法,对该方法的特异性、灵敏度等进行评价,并对多份临床样本进行应用检测。结果表明,该检测方法特异性强,对肠道病毒及其他人类非肠道病毒进行检测,显示了良好的特异性;该检测方法对EV71型和CA16型的检测灵敏度分别达到31.25 TCID50和1.25×102TCID50;将浓度为1×104TCID50及5×102TCID50的EV71样本进行重复性试验,其变异系数均小于1.5%;将浓度为5×102-5×105TCID50的EV71和CA16样本进行线性试验,其相关系数R2值在0.982-0.998之间。采用本研究建立的方法检测40份疑似临床样本,最后检出31份肠道病毒阳性样本,其中8例EV71型阳性,13例CA16阳性。另外,试验数据表明,内标对监控PCR抑制物的存在具有重要作用。本方法能同时快速检测所有肠道病毒并进行EV71型及CA16型的分型,并且灵敏度高、特异性好、扩增效率高,由于加入了内标,能有效地监控假阴性的出现,适合于手足口病的临床检测。