TaqMan-based Real-time RT-PCR Research Articles

Establishment of a duplex TaqMan-based real time RT-PCR assay for simultaneous detection of BRSV and BVDV

Bovine respiratory disease complex (BRDC) represents a global acute respiratory condition that imposes substantial economic burdens on the cattle industry due to its high morbidity and mortality rates. Various factors contribute to the development of BRDC, including pathogen infections, environmental stresses, weaning of calves, and herd relocation. Viral pathogens, notably bovine respiratory syncytial virus (BRSV) and bovine viral diarrhea virus (BVDV), play a critical role in the etiology of BRDC, with single or combined viral infections being particularly clinically significant. In this study, we developed a duplex TaqMan-based real-time RT-PCR assay targeting the conserved regions of the F gene of BRSV and the 5′ UTR sequence of BVDV. The limits of detection for BRSV and BVDV were 6.83 copies/μL and 5.24 copies/μL, respectively. Our validation data suggest the assay has excellent sensitivity, specificity and reproducibility. Testing of clinical samples revealed prevalence of BRSV and BVDV in local farms in Jiangsu Province, China. This study provides an efficient diagnostic tool for the epidemiological investigation of BRDC.

Association of a Genetic Variant in Chromosome 9p21 with Increased Risk of Developing Cervical Cancer

Background: Cervical cancer is one of the most prevalent gynecologic cancers associated with high morbidity and mortality worldwide. There is mounting evidence indicating an association between the 9p21 locus genetic variants with susceptibility to various human malignancies. In this current study, we aimed to evaluate the potential relationship between the rs1333049 genetic variant in chromosome 9p21 and the risk of cervical carcinogenesis. Methods: The possible correlation between rs1333049 polymorphism and susceptibility to cervical cancer was investigated in 221 patients with or without cancer. DNAs were isolated and genotyped using a TaqMan-based real-time RT-PCR method. Results: The rs1333049 genetic variant was found to be correlated with an elevated risk of cervical neoplasia using recessive and additive genetic models (p < 0.001). Conclusion: Our findings indicated that the CDKN2A/B genetic variant (rs1333049) was significantly associated with an elevated risk of cancer, suggesting its potential as a novel predictive marker for cervical carcinogenesis.

A nucleic acid detection assay combining reverse transcription recombinase-aided amplification with a lateral flow dipstick for the rapid visual detection of porcine deltacoronavirus

ABSTRACT Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen causing severe diarrhoea, dehydration, and death in nursing piglets and enormous economic losses for the global swine industry. Furthermore, it can infect multiple animal species including humans. Therefore, a rapid, definitive diagnostic assay is required for the effective control of this zoonotic pathogen. To identify PDCoV, we developed a nucleic acid detection assay combining reverse transcription recombinase-aided amplification (RT-RAA) with a lateral flow dipstick (LFD) targeting the highly conserved genomic region in the ORF1b gene. The RT-RAA-LFD assay exhibited good PDCoV detection reproducibility and repeatability and could be completed within 11 min. Ten minutes at 40 °C was required for nucleic acid amplification and 1 min at room temperature was needed for the visual LFD readout. The assay specifically detected PDCoV and did not cross-react with any other major swine pathogens. The 95% limit of detection (LOD) was 3.97 median tissue culture infectious dose PDCoV RNA per reaction. This performance was comparable to that of a reference TaqMan-based real-time RT-PCR (trRT-PCR) assay for PDCoV. Of 149 swine small intestine, rectal swab, and serum samples, 71 and 75 tested positive for PDCoV according to RT-RAA-LFD and trRT-PCR, respectively. The diagnostic coincidence rate for both assays was 97.32% (145/149) and the kappa value was 0.946 (p < 0.001). Overall, the RT-RAA-LFD assay is a user-friendly diagnostic tool that can rapidly and visually detect PDCoV.

Development of a new TaqMan-based real-time RT-PCR assay for the specific detection of bovine kobuvirus.

Bovine kobuvirus (BKV) is a novel kobuvirus considered to be closely related to calf diarrhea and has become a worldwide epidemic. Currently, the BKV lacks an efficient and convenient detection method to assist the research on BKV prevalence. In this study, a new and specific TaqMan-based real-time RT-PCR for the detection of BKV was developed using the conserved region of the 3D gene. The assay was highly specific for BKV, without cross-amplification with other non-targeted pathogens. The limit of detection of this assay was 102 copies. Standard curves showed a strong linear correlation from 102 to 106 copies of BKV standard RNA per reaction, and the parameters revealed as a slope of −3.54, efficiency of 91.64%, and regression coefficients (R2) of 0.998. The assay was also reproducible, with the intra-assay and inter-assay coefficient of variation <1.0%. The newly developed real-time RT-PCR was validated using 243 fecal samples collected from diarrheic or non-diarrheic cattle from nine regions in Hebei province and revealed the positive detection of BKV at a ratio of 19.34% (47/243). Sequencing of partial 3D genes from 13 positive samples and the following phylogenetic analysis demonstrated the reliability of the assay. In conclusion, the newly developed TaqMan-based real-time RT-PCR could be used for the screening and epidemic monitoring of BKV.

Development of TaqMan-based real-time RT-PCR assay based on N gene for the quantitative detection of feline morbillivirus

BackgroundMorbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is one of the members of Morbilliviruses that has been speculated to cause chronic kidney disease in cats even though a definite relationship is still unclear. To date, FeMV has been detected in several continents, such as Asia (Japan, China, Thailand, Malaysia), Europe (Italy, German, Turkey), Africa (South Africa), and South and North America (Brazil, Unites States). This study aims to develop a TaqMan real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection.ResultsA specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34–0.53% and 1.38–2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.ConclusionsThe TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.

Specific detection of the novel goose astrovirus using a TaqMan real-time RT-PCR technology

Recently, a novel goose astrovirus (N-GoAstV) was discovered in China, with the transmission route of N-GoAstV unclear. In this study, we developed a TaqMan-based real-time RT-PCR (qRT-PCR) assay for the detection of N-GoAstV infection. After the optimization of the qRT-PCR assay conditions, the results demonstrated that the lower limit of detection for N-GoAstV was 33.4 copies/μL. No cross-reactivity was observed with other goose-origin viruses. Intra-assay and inter-assay variability were ≤1.36% and 2.34%, respectively. N-GoAstV was detected in both field samples, embryos and newly hatched goslings by qRT-PCR assay, provided the view that N-GoAstV may be both horizontally and vertically transmitted. The established qRT-PCR method showed high specificity, sensitivity, and reproducibility, which can be used in future investigations on the pathogenesis and epidemiology of N-GoAstV.

Development of a TaqMan-based real-time RT-PCR assay for the detection of SADS-CoV associated with severe diarrhea disease in pigs

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus which was first reported in southern China in 2017. It can cause severe diarrhea disease in pigs. In order to detect this new emerging virus rapidly and reliably, a TaqMan-based real-time RT-PCR assay was established in this study. Specific primers and probe were designed and synthesized based on the conserved region within the N gene of the viral genome. Results showed that the lowest limit of detection was 3.0 × 101 copies/μL. This approach was specific for SADS-CoV, and there were no cross-reaction observed against other 15 swine viruses. It was 10 times more sensitive than the conventional PCR and gave higher SADS-CoV positive detection rate (70.69%, 123/174) than the conventional PCR (51.15%, 89/174) from clinical samples. These data indicated that the TaqMan-based real-time RT-PCR assay established here was an effective method with high sensitivity, specificity and reproducibility for faster and more accurate detection and quantification of SADS-CoV.

A survey of deformed wing virus and acute bee paralysis virus in honey bee colonies from serbia using real-time RT-PCR

Abstract In this study 55 honey bee colonies from different Serbian regions were monitored for the presence of Deformed Wing Virus (DWV) and Acute Bee Paralysis Virus (ABPV) using TaqMan-based real-time RT-PCR assay. The results revealed the presence of DWV in each sampling location, and ABPV in 10 out of 11 apiaries. High frequency of DWV (76.4%) and ABPV (61.8%) positive samples in asymptomatic colonies can be the consequence of inefficient and postponed Varroa treatment concerning the role of this mite in the transmission and activation of honey bee viruses. The real-time RTPCR technique described in this paper is proved to be the most reliable method for this kind of investigation.

Development and validation of a novel Taqman‐based real‐time RT‐PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is a serious disease in several fish species. VHS is caused by the rhabdovirus viral haemorrhagic septicaemia virus (VHSV). To prevent spreading of the pathogen, it is important to use a fast, robust, sensitive and specific diagnostic tool to identify the infected fish. Traditional diagnosis based on isolation in cell culture followed by identification using, for example, ELISA is sensitive and specific but slow. By switching to RT-PCR for surveillance and diagnosis of VHS the time needed before a correct diagnosis can be given will be considerably shortened and the need for maintaining expensive cell culture facilities reduced. Here we present the validation, according to OIE guidelines, of a sensitive and specific Taqman-based real-time RT-PCR. The assay detects all isolates in a panel of 79 VHSV isolates covering all known genotypes and subtypes, with amplification efficiencies of approximately 100%. The analytical and diagnostic specificity of the real-time RT-PCR is close to 1, and the analytical and diagnostic sensitivity is comparable with traditional cell-based methods. In conclusion, the presented real-time RT-PCR assay has the necessary qualities to be used as a VHSV surveillance tool on par with cell culture assays.

Diagnosis of Kyasanur forest disease by nested RT-PCR, real-time RT-PCR and IgM capture ELISA

Kyasanur forest disease (KFD) is a zoonotic viral disease caused by infection by a Flavivirus, a member of the family Flaviviridae. KFD is a public health concern in the Karnataka State in southern India. Available conventional diagnostic tests such as virus isolation and serological tests, such as haemagglutination inhibition and complement fixation tests are time consuming. This study reports the development of a nested RT-PCR [nRT-PCR] and a TaqMan-based real-time RT-PCR and IgM antibodies capture ELISA [MAC-ELISA] for rapid and accurate diagnosis of suspected KFD cases. The nRT-PCR and the TaqMan-based real-time RT-PCR assays were developed using gene sequences of the NS-5/non-coding region. Both the assays detected KFD viral RNA in acute phase human serum samples and can provide early diagnosis of infection. Real-time RT-PCR was found to be more sensitive than nRT-PCR, which could detect 38 copies of KFDV RNA. MAC-ELISA was developed for the detection of recent infections. Although real-time RT-PCR and nRT-PCR require expensive reagents, expensive equipment and trained personnel, the developed MAC-ELISA can be used easily in the affected areas. These tests add to the existing diagnosis arsenal against haemorrhagic viruses that are prevalent in India. These assays will also help to extend our knowledge of the pathology of KFD virus and its associated clinical features, by measuring the viral titre during infection and at the time of seroconversion. Information, which is not available currently because of the lack of appropriate diagnostic methods. In addition, early laboratory diagnosis of KFDV infection will help in the application of appropriate control measures and management of KFD cases.

Detection of five viruses infecting dormant bulbs by TaqMan-based real-time RT-PCR

Dormant bulbs may be infected by a number of viruses which are of potential economic importance for the horticultural industry. Five important viruses in bulbs were successfully detected by TaqMan-based real-time one-step RT-PCR assays: Arabis mosaic virus (ArMV), Cucumber mosaic virus (CMV), Lily symptomless virus (LSV), Tobacco rattle virus (TRV) and Tulip virus X (TVX). Nucleic acid of appropriate quality and quantity for RT-PCR was extracted from nine bulb species using the Qiagen RNeasy plant mini kit. Depending on the assay, the sensitivity of the real-time RT-PCRs varied from being the same sensitivity to 1,000 times more sensitive than the respective conventional RT-PCRs. The reliability of the TVX real-time RT-PCR was further assessed against conventional RT-PCR using leaves and bulbs of 24 Tulipa × hybrida plants. TVX was detected in twice the number of leaf samples than bulb samples for both conventional and real-time RT-PCRs.

Detection of genogroup IV norovirus in wastewater and river water in Japan

To test wastewater and river water in Japan for genogroup IV norovirus (GIV NoV). Influent and effluent samples from a wastewater treatment plant and the Tamagawa River water samples were collected monthly for a year. The water samples were concentrated by the adsorption-elution method, using an HA electronegative filter with acid rinse procedure, followed by quantitative detection of GIV NoV using TaqMan-based real-time RT-PCR. Both wastewater and river water samples showed a high positive ratio of GIV NoV during winter and spring. The highest concentration in wastewater and river water was 6.9 x 10(4) and 1.5 x 10(4) copies l(-1), respectively. Presence of GIV NoV in the environments demonstrates that not only GI and GII NoVs but also GIV strains are circulating and that routine monitoring of GIV NoV in water environments is recommended to understand its epidemics, environmental distribution and potential health risks. This is the first study providing quantitative data on the occurrence of GIV NoV in environmental water over a 1-year period.

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.

Melanoma vaccines: the paradox of T cell activation without clinical response.

In recent years significant progress in the understanding of the immune biology of melanoma has evolved from the identification of melanoma antigens (MAs) recognized by T cells. MAs consist of intracellular proteins that are expressed on the surface of cancer cells in association with human leukocyte antigen (HLA) class I molecules and therefore are suitable targets for cytotoxic T lymphocytes (CTLs). Several new monitoring strategies have been implemented to evaluate the status of activation and localization of vaccine-induced T cells in the peripheral circulation as well as the tumor site, including limiting dilution, in vitro sensitization, and ELISPOT. Previous studies aimed at monitoring patients receiving vaccination have utilized mainly those three methods. These methods have demonstrated that antigen-specific vaccination can elicit immune responses detectable in the peripheral blood of immunized patients. These assays, however, have been faulted by their requirement for in vitro expansion of T cells (limiting dilution or in vitro sensitization) or for limited sensitivity (ELISPOT). More recently, the use of soluble HLA/peptide complex tetramers, intracellular fluorescence-activated cell sorting (FACS) analysis, and real-time polymerase chain reaction (PCR) has been proposed for the monitoring of vaccine trials. These methods have the appeal of allowing direct enumeration of T cells specific for a particular epitope within relevant samples such as peripheral blood lymphocytes, lymph nodes, and tumors. We are evaluating whether utilizing a combination of HLA/peptide tetramer (tHLA) together with Taqman-based real-time reverse-transcription (RT)-PCR and intracellular FACS analysis could establish a direct and comprehensive strategy for the assessment of epitope-specific immune response in vivo. In conditions close to those of the tumor microenviroment or in peripheral blood lymphocytes, however, a different status of T cell activation can be expected due to a direct stimulation of T cells by tumor or antigen-presenting cells. We observed that activated T cells can easily be detected in the peripheral blood of patients who have received MA-specific vaccines. However, when T cells are stimulated with their relevant epitope, a high level of T cell receptor downregulation occurs that does not allow identification of vaccine-specific T cells directly with tHLA. Thus evaluation of epitope-specific T cells at the tumor site, where they might be exposed to stimulation by interaction with tumor cells and/or in bulk peripheral blood mononuclear cells, might be more efficiently analyzed with functional methods such as intracellular FACS and Taqman-based real time RT-PCR.