Development of a semi-quantitative real-time RT-PCR for the detection of measles virus.

Real-time detection of polymerase chain reactions allows convenient detection and quantification of virus-derived nucleic acids in clinical specimens. We have developed a real-time RT-PCR assay for the detection of measles virus (MV) genomic RNA, and compared it to a well-established conventional RT-PCR assay. Based on a serial dilution of the live-attenuated MV Edmonston Zagreb vaccine, the detection limits were approximately 0.1 and 0.02 cell culture infectious dose 50% units (CCID50) per test for the conventional and TaqMan RT-PCR assays, respectively. Furthermore, tissue materials spiked with known quantities of MV were equally well detected in both assays. The TaqMan assay was linear within a range of 10(4.4) to 10(-0.6)CCID50/ml, with an intra-assay variability lower than 3% and an inter-assay variability ranging from 1.5% at 10(4.4)CCID50/ml to 8.7% at 10(-0.6)CCID50/ml. The TaqMan assay could detect representative wild-type viruses from the currently active MV clades, and could detect MV genome in clinical specimens obtained from measles patients. Finally, quantification of MV RNA in peripheral blood mononuclear cells or broncho-alveolar lavage cells from cynomolgus macaques collected at different time points after experimental infection showed a good correlation with virus isolation data. In conclusion, the TaqMan assay developed is specific, sensitive, rapid and reproducible, and can be of use for diagnostic purposes or for studies on the pathogenesis of measles.     

Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control.

Two multiplex RT-PCR assays with specific coamplification of plant mRNA as an internal control from total nucleic acids are described for the parallel detection of Apple chlorotic leaf spot virus, Apple stem pitting virus, Apple mosaic virus and Apple stem grooving virus. All are important economically and common pathogens in commercial apple and pear cultivars, except for Apple mosaic virus. Four virus specific primer pairs and one primer pair which allows the specific amplification of mRNA of the mitochondrial nad5 gene are described. Specificity of all primer pairs was confirmed by sequencing the RT-PCR products. A range of different virus isolates from various geographic origins could be detected by these multiplex RT-PCR assays all year round. Viruses were detected reliably in composite extracts at a ratio of one part total nucleic acid extract from an infected sample mixed with 39 parts of extract from healthy samples. The use of the internal control minimizes the risk of obtaining false negative RT-PCR results, which is desirable for routine testing, and avoids the need to eliminate contaminating DNA in extracts. To our knowledge, this is the first report on the use of a specific internal RNA control from total nucleic acids. The multiplex RT-PCR assays described are reliable, rapid and sensitive methods for the detection of these viruses, and may replace techniques need commonly like indexing by woody indicators or ELISA.     

Taqman荧光定量RT—PCR在呼吸道感染患者临床标本非SARS冠状病毒检测中的应用

目的建立检测四种常见人非SARS冠状病毒核酸特异的快速、敏感的TaqManqRT—PCR检测方法,应用于急性呼吸道感染患儿的感染分析。方法分别应用TaqManqRT—PCR与普通RT—PCR平行检测248份呼吸道标本,对方法的灵敏性、特异性和稳定性以及临床标本的适用性进行比较评价,阳性标本以体外转录RNA为标准品进行病毒载量定量。结果本方法可对HKU1、NL63、229E、OC43四种冠状病毒进行特异性诊断,与其他病毒无交叉反应,检测灵敏度可达10拷贝／μl,检测线性范围可达10^1～10^8拷贝／μl,248份标本中HKU1、NL63、229E、OC43阳性率依次为1．2％,0．8％,1．2％,1．6％,其中OC43荧光RT—PCR法检出率高于普通RT—PCR,其余三种病毒两种方法检测结果一致。非SARS冠状病毒阳性标本均检出于12月至次年5月。结论建立的TaqManRealtimeRT-PCR法具有特异性强、灵敏性高的特点,是开展非SARS冠状病毒的临床检测与疾病监测的有效技术手段。     

Development and validation of a TaqMan real-time reverse transcription-PCR for rapid detection of feline calicivirus

Feline calicivirus (FCV) is a common cause of upper respiratory tract disease in cats and is associated with interstitial pneumonia, oral ulceration and polyarthritis. Recently, outbreaks have involved a highly virulent FCV that leads to multisystemic signs. Virus isolation and conventional RT-PCR are the most common methods used for FCV diagnosis. However, real-time RT-PCR offers a rapid, sensitive, specific and easy tool for nucleic acid detection. The objective of this study was to design a TaqMan probe-based, real-time RT-PCR assay for detection of FCV. It was determined in our previous study that the first 120 nucleotides of the 5′ region of the genome are highly conserved among FCV isolates. Primers and a probe specific for this region were designed for a real-time RT-PCR assay to detect FCV. Initial validation was done using 15 genetically diverse isolates. Also, 122 samples were tested by the new assay and virus isolation. The real-time RT-PCR assay was as sensitive and specific as virus isolation and was far more rapid. This real-time RT-PCR assay targeting the conserved 5′ region of the genome is a fast, economical and accurate method for detection of FCV.     

Biotinylated nucleic acid hybridization probes for potato virus detection

Summary cDNA libraries, representative of potato viruses X (PVXc strain) and Y (PVY^o strain) genomes were obtained. A PVX cDNA cloned fragment was sequenced and biotinylated to be used as hybridization probe for the detection of purified virus or nucleic acid extracts of infected plants. Dot hybridization assay was sensitive to detect 4 ng of viral particles, corresponding to about 200 pg of viral RNA. The level of detection in infected plant extracts was as effective as that obtained with the ELISA. The presence of biotinylated PVY cDNA in the hybridization mixture did not affect sensitivity of the PVX detection assay, suggesting that a single diagnostic assay for several potato viruses and virus-related pathogens could be developed.     

Detection of Colorado Tick Fever viral RNA in acute human serum samples by a quantitative real-time RT-PCR assay

A quantitative real-time RT-PCR assay for the detection of Colorado Tick Fever (CTF) viral RNA in human clinical samples is presented. The sensitivity of this assay has been shown to be greater than that of the isolation of virus in Vero cells by standard plaque assay in a direct comparison. The specificity of the CTF quantitative real-time RT-PCR assay was determined by the exclusive detection of CTF viral RNAs when applied to a diverse panel of CTF viral isolates and reference strain agents known to circulate in areas of CTF virus transmission. Lastly, the quantitative real-time RT-PCR assay demonstrated exceptional sensitivity for the detection of CTF viral RNA in acute human serum. The quantitative real-time RT-PCR assay is efficient, sensitive and specific and as such is useful for the detection of CTF viral RNA in the diagnostic or research laboratory.     

Comparison of low-density arrays, RT-PCR and real-time TaqMan RT-PCR in detection of grapevine viruses

Low-density arrays (LDA) have been designed based on the real-time RT-PCR (TaqMan) assays for the specific detection of 13 viruses that infect Grapevines in addition to the housekeeping gene 18S rRNA. The viruses included in the study are Grapevine leafroll associated viruses 1, 2, 3, 4, 5, and 9, Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG) strain, Ruspestris stem pitting associated virus, Grapevine vitivirus A, Grapevine vitivirus B, Grapevine fanleaf virus, Tomato ringspot virus (ToRSV), and Grapevine fleck virus (GFkV). This study includes three new TaqMan RT-PCR assays that have been developed for GLRaV-2RG, GFkV and ToRSV and have been included in the TaqMan RT-PCR and LDA detection. The LDAs were evaluated against a wide range of isolates distributed geographically. Geographical locations included Africa, Europe, Australia, Asia, Latin America and the United States. High-throughput detection of these viruses using LDAs was compared to RT-PCR and real-time TaqMan RT-PCR. The efficiency of different RNA extraction methodologies and buffers were compared for use in low-density array detection. In addition improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan assay as an RNA specific internal control proved to generate better diagnostic assays. This is the first report on the use of LDA for the detection of plant viruses.