A sensitive one-step real-time RT-PCR method for detection of deformed wing virus and black queen cell virus in honeybee Apis mellifera

A one-step real-time RT-PCR based on SYBR Green (SG) chemistry was developed for the detection, differentiation and quantification of two of the most common viruses on the honeybee Apis mellifera L., deformed wing virus and black queen cell virus. Two sets of primers specific for each virus, were designed in conserved regions of the viral genome for their use in the one-step real-time RT-PCR. Both reactions were optimized for highest sensitivity and specificity and SG-based real-time was used to achieve quantitative detection. All samples evaluated in this study were from Spanish honeybee colonies. Viral detection and identification was confirmed by sequencing of the PCR products. The described one-step real-time SG RT-PCR proved to be a fast, accurate and useful technique to detect and even quantify these honeybee viruses that cause unapparent infections, and might contribute with other factors to the increasing honeybee colonies depopulation.     

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.     

A sensitive one-step real-time RT-PCR method for detecting Grapevine leafroll-associated virus 2 variants in grapevine

Grapevine leafroll syndrome is caused by a complex of up to nine different Grapevine leafroll-associated viruses (GLRaV-1-9) with GLRaV-2 being reported as one of the most variable species of this group. Many methods, including indexing, serological and molecular procedures, have been developed for the detection of GLRaV-2. However, due to the low concentration of the virus in plants and the high variability of GLRaV-2, a method with improved sensitivity and with the capacity to detect of all known variants is required. Such improvement is essential for grapevine rootstocks, as these are suspected to harbour frequent GLRaV-2 infections difficult to detect, thus contributing to the spread of the leafroll disease. The development of new universal primers is described using a target sequence located in the 3' end of the virus genome. These primers were combined with a one-step SYBR Green real-time RT-PCR assay to achieve quantitative detection. All 43 GLRaV-2 isolates tested in this study were identified readily and reproducibly, regardless of their geographical origin or variety of grapevine. Using the procedure developed in this study, the sensitivity was increased 125 times compared to a conventional single-tube RT-PCR. This real-time method opens new perspectives for the sanitary selection of grapevine and in leafroll 2 disease monitoring.     

Development of a minor groove binder assay for real-time one-step RT-PCR detection of swine vesicular disease virus

The design and development of a 5′ conjugated minor groove binder (MGB) probe real-time RT-PCR assay are described for rapid, sensitive and specific detection of swine vesicular disease virus (SVDV) RNA. The assay is designed to target the 2C gene of the SVDV genome and is capable of detecting 2 × 10² copies of an RNA standard per reaction. It does not detect any of the other RNA viruses that cause vesicular disease in pigs, or the human enterovirus, Coxsackie B5 virus (CVB5) which is closely related antigenically to SVDV. The linear range of this test was from 2 × 10² to 2 × 10⁸ copies/μl. The assay is rapid and can detect SVDV RNA in just over 3.5 h including the time required for nucleic acid extraction. The development of this assay provides a useful tool for the differential diagnosis of SVD or for the detection of SVDV in research applications. This study demonstrates the suitability of MGB probes as a real-time PCR chemistry for the diagnosis of swine vesicular disease.     

Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus

One-step SYBR Green I-based real-time RT-PCR assay for rapid detection as well as quantitation of Japanese encephalitis virus (JEV) in acute-phase patient CSF samples by targeting the NS3 gene was developed. The assay developed in this study was found to be more sensitive as compared to conventional RT-PCR. The specificity of the reported assay system was established through melting curve analysis as well as by cross-reactivity studies with related members of Flavivirus. The applicability of Real-time PCR assay for clinical diagnosis was validated with 32 suspected acute-phase CSF samples of Gorakhpur epidemic, India, 2005. The improved sensitivity of real-time RT-PCR was reflected by picking up 10 additional samples with low copy number of template in comparison to conventional RT-PCR. The quantitation of the viral load in acute-phase CSF samples was done using a standard curve obtained by plotting cycle threshold (C(t)) values versus copy numbers of the RNA template. This is the first report on the application of real-time RT-PCR for detection as well as quantitation of JEV from patient CSF samples. These findings demonstrate the potential clinical application of the reported assay as a sensitive diagnostic test for rapid and real-time detection and quantitation of JEV in acute-phase clinical samples.     

Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of Chikungunya virus.

The development of a one-step SYBR Green I-based real-time RT-PCR assay is reported for detection and quantification of Chikungunya virus (CHIKV) in acute-phase patient serum samples by targeting the E1 structural gene. A linear relationship was obtained between the virus concentration and cycle threshold (C(t)) value over a range of 10(7)-0.1PFU/ml. The reported assay was found to be 10-fold more sensitive compared to conventional RT-PCR with a detection limit of 0.1PFU/ml. The feasibility of this reported assay system for clinical diagnosis was validated with 51 suspected acute-phase serum samples of the recent CHIKV epidemic in southern India, 2006. The comparative evaluation with acute-phase patient serum samples revealed the higher sensitivity of real-time RT-PCR assay by picking up six additional samples with low copy number of template. None of the healthy serum samples analyzed in this study showed amplification. The quantification of the viral load in the acute-phase serum samples was also determined employing the standard curve, which varies from 0.1 to 10(7)PFU/ml. These findings demonstrated that the reported assay has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of Chikungunya virus in acute-phase patient serum samples.     

Development of a one-step real-time RT-PCR assay using a minor-groove-binding probe for the detection of duck Tembusu virus

The design and development of a 3'-conjugated minor-groove-binding (MGB) probe for a real-time RT-PCR assay allowing for the rapid, sensitive, and specific detection of duck Tembusu virus (DTMUV) RNA are described. This assay targeted the 3' terminal non-coding region (NCR) of the TMUV genome and detected 1 × 101 copies of RNA per reaction without cross-reaction with other duck pathogens. The linear range of detection was 2 × 101-2 × 10? copies/μl. The assay was rapid, requiring just over 2.0 h, including the nucleic acid extraction step. Therefore, this assay is an excellent tool for research routine diagnostic applications, and study of the epidemiology of TMUV infections among duck flocks.     

Comparison of real-time fluorescent RT-PCR and conventional RT-PCR for the detection of hepatitis E virus genotypes prevalent in China

To compare the specificity and sensitivity of a real-time fluorescent RT-PCR assay with conventional RT-PCR, sera from 110 healthy blood donors, 120 patients with a clinical diagnosis of chronic hepatitis B, and 416 patients with non-A-C acute hepatitis, as well as serial dilutions of HEV genotypes 1 and 4, were tested with both assays. All samples from healthy blood donors and patients with chronic hepatitis B were negative by both assays. Real-time RT-PCR could detect the same final dilution of genotype 1 as conventional RT-PCR but could detect a 10-fold lower concentration of genotype 4 than conventional RT-PCR. Of 416 samples from patients with a clinical diagnosis of non-A-C acute hepatitis, 127 (30.5%) and 83 (20.0%) were positive for HEV by real-time and conventional RT-PCR, respectively. The concordance of real-time and conventional RT-PCR was 80.8%. Furthermore, 96 and 57 of 171 samples were positive for anti-HEV IgM by real-time and conventional RT-PCR, respectively, and 31 and 26 of 245 samples negative for anti-HEV IgM, were positive by real-time and conventional RT-PCR, respectively. All amplicons positive by conventional RT-PCR were sequenced. Of 83 isolates, 7 and 76 belonged to genotypes 1 and 4, respectively. Thus, both assays have a high specificity, but the real-time RT-PCR assay is more sensitive than conventional RT-PCR. Furthermore, HEV genotype 4 is responsible for most sporadic cases of hepatitis E in the north of China.     

A simple one-step real-time RT-PCR for diagnosis of dengue virus infection

Dengue is the most important arbovirus disease in tropical and sub-tropical countries, and can be caused by infection with any of the four-dengue virus (DENV) serotypes. Infection with DENV can lead to a broad clinical spectrum, ranging from sub-clinical infection or an influenza-like disease known as dengue fever (DF) to a severe, sometimes fatal, disease characterized by hemorrhage and plasma leakage that can lead to shock, known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The diagnosis of dengue is routinely accomplished by serologic assays, such as IgM and IgG ELISAs, as well as HI tests, analyzing serum samples obtained from patients with at least 7 days of symptoms onset. These tests cannot be used for diagnosis during the early symptomatic phase. In addition, antibodies against dengue are broad reactive with other flaviviruses. Therefore, a specific diagnostic method for acute DENV infection is of great interest. In that sense, the real-time RT-PCR has become an important tool that can be used for early and specific detection of dengue virus genome in human serum samples. This study describes a simple, specific, and sensitive real-time RT-PCR for early diagnosis of dengue virus infection.     

Development and evaluation of a real-time RT-PCR assay for Sindbis virus detection

Sindbis virus (SINV) is an arthropod-borne alphavirus found widely in Eurasia, Africa and Oceania. Clinical SINV infection, characterized by rash and arthritis, is reported primarily in Northern Europe. The laboratory diagnosis of SINV infection is based currently on serology. A one-step TaqMan^® real-time RT-PCR assay was developed for the detection of SINV and evaluated its clinical performance with acute-phase serum samples. The specificity and sensitivity of the real-time PCR assay were assessed using cell cultured Finnish SINV strains. The applicability of the assay for diagnostic use was evaluated using 58 serum samples from patients infected with SINV. The real-time RT-PCR assay was specific and sensitive for the detection of SINV in cell culture supernatants with a 95% detection limit of 9 genome copies/reaction determined by probit analysis. However, in the assay only 7/58 (12%) of serum samples were positive of which two were also positive by conventional nested PCR assay and none by virus isolation. This novel assay is specific and sensitive for detection of SINV and can be used for example for screening SINV in wildlife. However, molecular diagnostic techniques using serum samples seem to be of limited value for the diagnosis of human SINV infection due to the short and low viraemia of infection with SINV.     

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.     

Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma

Dengue is mosquito-borne virus infection that annually causes ∼50 million clinically apparent cases worldwide. An internally controlled one-step real-time multiplex RT-PCR assay was developed for detection and quantitation of DENV RNA in plasma sample by using specific primers and fluorogenic TaqMan probes. All primers and probes targeted sequences near the 3′ end of the NS5 gene. The method comprised two multiplex assays and was validated for sensitivity, specificity, linearity, reproducibility and precision. An internal control template was spiked into each clinical specimen to provide quality assurance for each experimental step. The assay allowed for detection of between 0.5 and 3 infectious particles per mL, is rapid and has been operationally characterized in 287 Vietnamese dengue patients from two therapeutic intervention trials at the Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam.