Molecular characterization of two Pepino mosaic virus variants from imported tomato seed reveals high levels of sequence identity between Chilean and US isolates

Pepino mosaic virus (PepMV), a member of the genus Potexvirus, was first described in South America on pepino (Solanum muricatum A.). Only in recent years, it was reported to infect greenhouse-grown tomatoes. Genome nucleotide sequences from several European isolates showed extensive sequence identity (>99%). Recent genome nucleotide sequences from two US isolates (US1 and US2) however showed much greater sequence divergence from that of the European PepMV isolates. My interest in characterizing virus isolates from South America was due to an active commercial tomato seed production in Chile. Through genome sequence comparison and phylogenetic analyses, we may be able to understand the source of virus infection and control this devastating disease from further spreading into new tomato growing regions of the world. Complete genome nucleotide sequences from two PepMV variants (designated as Ch1 and Ch2) were determined from a virus isolate obtained from a commercial tomato seed lot produced in Chile. Using RT-PCR-based genome walking strategy, complete genome sequences from these two variants were determined. Excluding poly (A) tails, the genomes of PepMV Ch1 and Ch2 were 6414 and 6412 nucleotides (nt), respectively. Pairwise comparisons of PepMV Ch1 and Ch2 genomes with other PepMV isolates showed that the highest nucleotide sequence identity was with two US isolates, 98.7% between PepMV Ch1 and US1, and 90.7% between Ch2 and US2. Similar to PepMV US1 and US2, the two Chilean variants were the most divergent from one another (78% nt identity). These two Chilean PepMV variants also shared only 78–86% nucleotide sequence identity to that of five European isolates. The high level of nucleotide sequence identity between Chilean and US isolates suggests a common origin. Phylogenetic analyses with various gene products generated three distinct sequence clusters (or strains): US1 and Ch1 in the first group, US2 and Ch2 in the second, and the European tomato isolates in the third. Based on the host specificity, it was previously suggested that the original pepino isolate should be considered a distinct strain from that of the tomato isolates. Nucleotide sequence data reported in this paper were submitted to the GenBank database and given the accession numbers DQ000984 for PepMV Ch1 and DQ000985 for PepMV Ch2. The use of trade, firm, or corporation names in this article does not imply the endorsement or approval by the USDA, ARS of any product to the exclusion of others that may be suitable.     

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。     

H9N2禽流感病毒一步法双重荧光RT-PCR检测方法的建立

目的 建立一种可同时检测禽流感病毒H9N2的HA和NA基因一步法双重荧光RT-PCR方法.方法 针对H9N2禽流感病毒的HA和NA基因保守区,设计相应的特异性引物以及探针,优化检测体系及反应条件,建立一步法双重荧光定量RT-PCR方法.对该方法的灵敏度、特异性、稳定性进行验证与评估,并对家禽粪便标本进行应用检测,以单重实时荧光RT-PCR方法作为参照,检测结果不一致的样本采用测序进行验证.结果 该方法特异性强,与H1、H3、H5、H7亚型禽流感病毒、鸡新城疫及鸡传染性支炎病毒均无交叉反应,对HA和NA基因的最低检出限分别可达50拷贝/μL和25拷贝/μL,组间与组内的变异系数在0.20 ～0.79％之间.对82份粪便标本进行检测,H9N2禽流感病毒的阳性率为8.14％ (7/82),与单重实时荧光RT-PCR法检测结果一致.结论 该方法特异性强、灵敏度高、稳定性好,可应用于临床禽流感样本的检测.     

Rapid detection, serotyping and quantitation of dengue viruses by TaqMan real-time one-step RT-PCR

The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.     

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.     

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.     

Quantitative real-time PCR for detection of monkey B virus (Cercopithecine herpesvirus 1) in clinical samples

A TaqMan based real-time PCR assay was developed for rapid detection and quantitation of herpes B virus (Cercopithecine herpesvirus 1) in clinical samples. The assay utilizes B virus-specific primers and a probe to the non-conserved region of the gG gene to discriminate B virus from closely related alphaherpesviruses. Fifty copies of B virus DNA could be detected with 100% sensitivity with a wide range of quantitation spanning 6 logs. The assay was highly reproducible with intra- and inter-assay coefficients of variation of 0.6 and 2.4%, respectively. Clinical utility of the developed real-time PCR was evaluated by testing genomic DNA prepared from B virus clinical isolates (n=23) and human and monkey clinical specimens (n=62). This novel method was also compared with conventional cell culture with respect to sensitivity and specificity. TaqMan PCR assay was shown to be equally specific and more sensitive than culture method (culture vs. PCR sensitivity 50%) and was able to identify all B virus clinical isolates tested. Fast, reliable assessment of B virus DNA in infected cells and tissues makes real-time PCR assay a valuable tool for diagnosis and management of B virus infections.     

产毒型O1群霍乱弧菌特异性基因序列实时荧光双重TaqMan PCR快速检测体系的建立

目的建立针对O1群霍乱弧菌的高敏感、高特异的实时荧光双重TaqMan聚合酶链式反应快速检测体系。 方法根据O1群霍乱弧菌主基因组O抗原编码基因rfb-O1和霍乱肠毒素的A亚基编码基因ctxA的特异性序列设计引物及TaqMan探针,利用便携式Smartcycler Ⅱ实时荧光PCR检测平台探讨该检测体系的敏感性,用19种其他肠道致病菌及院内感染常见致病菌评价该检测体系的特异性。 结果实时荧光双重TaqMan聚合酶链式反应快速检测体系对O1群霍乱弧菌的检测敏感度为1．0×10^2拷贝每反应体系;对O1群霍乱弧菌基因组DNA的检测敏感度为1．0×10^1 pg每反应体系;该检测体系在检测19种其他肠道致病菌或院内感染中的常见致病菌时不存在非特异性扩增;整个反应在2h内完成。 结论本研究建立的实时荧光双重TaqMan聚合酶链式反应检测体系可作为产毒型O1群霍乱弧菌特异、敏感、快速的检测手段,也可同时检测O1群霍乱弧菌产生霍乱肠毒素的能力。     

Rapid differential detection of classical and highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus in China by a duplex real-time RT-PCR

Since the emergence of highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus (H-US-PRRSV) in 2006, the classical North American PRRSV (C-US-PRRSV) and H-US-PRRSV isolates have coexisted in Chinese swine herds. A duplex real-time RT-PCR assay using minor groove binder (MGB) probes for differential detection of the two US PRRSV isolates was developed. The specificity, sensitivity, reproducibility, and interference test of this assay were validated. The sensitivity of the assay was 3.2 TCID₅₀/ml or 38 RNA copies/μl for C-US-PRRSV and 0.4 TCID₅₀/ml or 14 RNA copies/μl for H-US-PRRSV. Both assays were 10 times more sensitive than the current methods. A total of 302 clinical samples were tested by duplex real-time RT-PCR and conventional RT-PCR assays, and the results showed over 98.7% agreement. In addition, the new assay can be completed in less than 2 h. This duplex real-time RT-PCR assay is a promising tool for rapid differential detection and epidemiology of US PRRSV in China.     

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。     

Comparative evaluation of new TaqMan real-time assays for the detection of hepatitis A virus

Three novel real-time TaqMan RT-PCR assays targeting the 5′-UTR, the viral protease and the viral polymerase regions of the hepatitis A virus (HAV) were developed, evaluated and compared against a new published 5′-UTR TaqMan assay (JN) and a widely used conventional RT-PCR assay (HAVc). All conventional RT-PCR (HAV, SH-Prot and SH-Poly systems) and TaqMan (SH-Prot, SH-Poly, JN and SH-5U systems) assays evaluated were consistent for the detection of the three different HAV strains (HM-175, HAS-15 and LSH/S) used and reproducible for both RNA duplicates with the exception of two reproducibility discrepancies observed with both 5′-UTR real-time systems (JN and SH-5U). Limits of detection for conventional HAV, SH-Prot and SH-Poly RT-PCR systems were found to be equivalent when tested with serially diluted suspensions of the HM-175 strain. Although the four real-time RT-PCR TaqMan assays evaluated herein produced similar and consistent quantification data down to the level of one genomic equivalent copy with their respectively cloned amplicons, significant and important differences were observed for the detection of HAV genomic RNA. Results showed that the new real-time TaqMan SH-Poly and SH-Prot primer and probe systems were more consistent and sensitive by 5 logs as compared to both 5′-UTR designs (JN and SH-5U) used for the detection of HAV genomic RNA as well as for the detection in cell culture by cytopathic effect. Considering their higher analytical sensitivity, the proposed SH-Poly and SH-Prot amplification systems could therefore represent valuable tools for the detection of HAV in clinical, environmental and food samples.     

Rapid detection of enterovirus 71 by real-time TaqMan RT-PCR.

BACKGROUND: Enterovirus 71 (EV71) is the main etiological agent of Hand, Foot and Mouth Disease (HFMD) and has been associated with neurological complications which resulted in fatalities during recent outbreaks in Asia Pacific region. OBJECTIVE: Develop a real-time TaqMan RT-PCR for rapid detection of EV71. STUDY DESIGN: Specific primers and probe were designed based on highly conserved VP1 region of EV71. The sensitivity of the real-time RT-PCR was evaluated with 67 clinical specimens collected from pediatric patients with suspected HFMD. RESULTS: Our real-time TaqMan RT-PCR showed 100% specificity in detecting EV71 and showed an analytical sensitivity of 5 viral copies. High sensitivity was also achieved in detecting EV71 directly from clinical specimens. CONCLUSIONS: Real-time TaqMan RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks.     

Development and validation of a TaqMan-MGB real-time RT-PCR assay for simultaneous detection and characterization of infectious bursal disease virus

Rapid and reliable detection and classification of infectious bursal disease viruses (IBDVs) is of crucial importance for disease surveillance and control. This study presents the development and validation of a real-time RT-PCR assay to detect and discriminate very virulent (vv) from non-vv (classic and variant) IBDV strains. The assay uses two fluorogenic, minor groove-binding (MGB) TaqMan probes targeted to a single nucleotide polymorphism (SNP) embedded in a highly conserved genomic region. The analytical sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA. The assay demonstrated a wide dynamic range between 10(2) and 10(8) standard RNA copies per reaction. Good reproducibility was also detected, with intra- and inter-assay coefficients of variation ranging from 0.13% to 2.23% and 0.26% to 1.92%, respectively. The assay detected successfully all the assessed vv, classical, and variant field and vaccine strains and correctly discriminated all vvIBDV strains from non-vvIBDV strains. Other common avian RNA viruses tested negative, indicating high specificity of the assay. The high sensitivity, rapidity, reproducibility, and specificity of the real-time RT-PCR assay make this method suitable for general and genotype-specific detection and quantitation.