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 SYBR Green I-based real-time PCR assay for detection of drug resistance mutations in cytomegalovirus

Ganciclovir (GCV) is an antiviral drug that is used to treat cytomegalovirus (CMV) infection. However, long-term monotherapy does not commonly result in complete suppression of viral replication and is associated with the emergence of resistant mutants. In this study, a method for detecting CMV resistance mutations was carried out by real-time amplification refractory mutation system PCR (real-time ARMS PCR) using SYBR Green I fluorescent dye. Three recombinant plasmids were constructed by overlapping extension PCR to be used as standard mutation or wild-type models. Four pairs of primers were used to amplify the approximately 150 bp of the UL97 gene spanning codon 460, where mutations associated with resistance to GCV invariably occur. As little as 20% mutants DNA in 10(7)copies/ml mixture DNA were detected. Though this approach was not more sensitive than PCR-restriction fragment length polymorphism (RFLP) for the detection of the presence of mixtures, it was a high-throughput and automation method, and the specific mutation type can be deduced by the real-time ARMS PCR data. Overall, this study has demonstrated an approach that could be a sensitive and rapid method for the detection of GCV resistance-associated mutation in CMV.     

Development of a real-time RT-PCR assay for detection and quantitation of parainfluenza virus 3

A TaqMan-based real-time RT-PCR assay was developed to detect and quantify human parainfluenza virus 3 (PIV3). Two sets of primer–probe pairs were designed based on the nucleotide (nt) sequence of the nucleocapsid (N) gene. The primer–probe pairs were derived from the 3′ end of the N gene (set 1) and the 5′ region of the gene (set 2), respectively. Using real-time RT-PCR, the sensitivity of set 1 was determined to be about 9 copies of PIV3 genome, while the sensitivity of set 2 was about 93 copies of PIV3 genome. Set 1 was chosen for subsequent experiments. This primer–probe pair detected PIV3, but not any of several other respiratory viruses, indicating that the assay is PIV3 specific. For clinical evaluation, the assay was employed to test 80 nasopharyngeal aspirates from children with respiratory symptoms. The results confirmed the presence of PIV3 in 12 specimens previously identified as positive by culture confirmation, and showed all of which contained more than 100 copies of PIV3 genome. In addition, the method also detected PIV3 genomes in specimens found negative by culture confirmation, indicating the value of this RT-PCR assay. These data thus demonstrate the application of the real-time RT-PCR assay for the detection and quantification of PIV3 in clinical specimens.     

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.     

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.     

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 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.     

A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus

Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic forms of acute hepatitis. Large waterborne HEV epidemics have been documented exclusively in developing countries. At least four major genotypes of HEV have been reported worldwide: genotype 1 (found primarily in Asian countries), genotype 2 (isolated from a single outbreak in Mexico), genotype 3 (identified in swine and humans in the United States and many other countries), and genotype 4 (identified in humans, swine and other animals in Asia). To better detect and quantitate different HEV strains that may be present in clinical and environmental samples, we developed a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA. Primers and probes for the real-time RT-PCR were selected based on the multiple sequence alignments of 27 sequences of the ORF3 region. Thirteen HEV isolates representing genotypes 1-4 were used to standardize the real-time RT-PCR assay. The TaqMan assay detected as few as four genome equivalent (GE) copies of HEV plasmid DNA and detected as low as 0.12 50% pig infectious dose (PID50) of swine HEV. Different concentrations of swine HEV (120-1.2PID50) spiked into a surface water concentrate were detected in the real-time RT-PCR assay. This is the first reporting of a broadly reactive TaqMan RT-PCR assay for the detection of HEV in clinical and environmental samples.     

Development of TaqMan real-time PCR assay for detection and quantitation of reticuloendotheliosis virus

A highly sensitive real-time PCR method was developed in this study for reticuloendotheliosis virus (REV) detection and quantitation. The real-time PCR method, with a minimum detection limit of 10 proviral DNA copies, was 100 times more sensitive than the conventional PCR. It was also shown to be highly specific, as no positive signals were detected for other common avian DNA viruses. The coefficients of variation of intra- and inter-assay reproducibility were both less than 2%. The chicken β-actin gene was co-amplified and used as the internal control to monitor the efficiency of DNA extraction and PCR amplification. Specific pathogen free chickens were infected with REV at different ages and the blood was detected with the real-time PCR method. High levels of proviral DNA were detected in the blood of REV-infected chickens during the experiment and chickens infected early had higher proviral loads from 2 weeks post-infection compared with late infected chickens. This study provides an excellent research and diagnostic tool that can be used for REV detection and quantitation.     

Development and evaluation of a one-step real-time RT-PCR assay for universal detection of influenza A viruses from avian and mammal species

The objective of our study was to develop and evaluate a TaqMan real-time RT-PCR (RRT-PCR) assay for universal detection of influenza A (IA) viruses. The primers and LNA-modified octanucleotide probe were selected to correspond to extremely conserved regions of the membrane protein (MP) segment identified by a comprehensive bioinformatics analysis including 10,405 IA viruses MP sequences, i.e., all of the sequences of the Influenza Virus Sequence database collected as of August 20, 2009. The RRT-PCR has a detection limit of approximately five copies of target RNA/reaction and excellent reaction parameters tested in four IA viruses reference laboratories. The inclusivity of the assay was estimated at both the bioinformatic and the experimental level. Our results predicted that this RRT-PCR assay was able to detect 99.5% of known human IA virus strains, 99.84% of pandemic influenza A (H1N1) strains, 99.75% of avian strains, 98.89% of swine strains, 98.15% of equine strains, and 100% of influenza A viruses of other origin.     

A SYBR Green I real-time RT-PCR assay for detection and differentiation of influenza A(H1N1) virus in swine populations

The novel influenza A(H1N1) virus that emerged recently in Mexico has spread rapidly to many countries and initiated a human pandemic. It would be interesting to determine whether the virus has existed in, or will spread to, the swine population. However, it is difficult to differentiate the virus from some swine influenza viruses. In this study, a SYBR Green I real-time RT-PCR assay was designed for detection and differentiation of influenza A(H1N1) virus from some swine influenza viruses, by comparing the amplification of two pairs of primers corresponding to influenza A(H1N1) virus and some swine influenza viruses, respectively. The assay was evaluated using online analysis, identified influenza viruses and clinical samples. The results indicated that the assay has high sensitivity and specificity to detect influenza A(H1N1) virus, and is able to differentiate it from some swine influenza viruses. This, in turn, could provide essential epidemiological information for risk analysis and decision making in combating the disease, and stimulate research to differentiate pathogens similar to each other using the same method.