scholarly journals Evaluation of a Trio Toscana Virus Real-Time RT-PCR Assay Targeting Three Genomic Regions within Nucleoprotein Gene

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 254
Author(s):  
Laurence THIRION ◽  
Laura PEZZI ◽  
Irene PEDROSA-CORRAL ◽  
Sara SANBONMATSU-GAMEZ ◽  
Xavier de de LAMBALLERIE ◽  
...  
Keyword(s):  
Real Time ◽  
Qpcr Assay ◽  
Clinical Samples ◽  
Toscana Virus ◽  
Nucleoprotein Gene ◽  
Specificity And Sensitivity ◽  
Mediterranean Countries ◽  
Rna Targeting ◽  
Genomic Regions ◽  
Better Than

Toscana virus (TOSV) can cause central nervous system infections in both residents of and travelers to Mediterranean countries. Data mining identified three real-time RT-qPCR assays for detecting TOSV RNA targeting non-overlapping regions in the nucleoprotein gene. Here, they were combined to create a multi-region assay named Trio TOSV RT-qPCR consisting of six primers and three probes. In this study, (i) we evaluated in silico the three RT-qPCR assays available in the literature for TOSV detection, (ii) we combined the three systems to create the Trio TOSV RT-qPCR, (iii) we assessed the specificity and sensitivity of the three monoplex assays versus the Trio TOSV RT-qPCR assay, and (iv) we compared the performance of the Trio TOSV RT-qPCR assay with one of the reference monoplex assays on clinical samples. In conclusion, the Trio TOSV RT-qPCR assay performs equally or better than the three monoplex assays; therefore, it provides a robust assay that can be used for both research and diagnostic purposes.

scholarly journals A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

2020 ◽  
Vol 21 (8) ◽  
pp. 2826 ◽  
Author(s):  
Renfei Lu ◽  
Xiuming Wu ◽  
Zhenzhou Wan ◽  
Yingxue Li ◽  
Xia Jin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
N Gene ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

scholarly journals Human and entomological surveillance of Toscana virus in the Emilia-Romagna region, Italy, 2010 to 2012

2014 ◽  
Vol 19 (48) ◽  
Author(s):  
M Calzolari ◽  
P Angelini ◽  
A C Finarelli ◽  
R Cagarelli ◽  
R Bellini ◽  
...  
Keyword(s):  
Interdisciplinary Approach ◽  
Viral Meningitis ◽  
Integrated System ◽  
Clinical Samples ◽  
Toscana Virus ◽  
Entomological Surveillance ◽  
Mediterranean Countries ◽  
Phlebotomine Sandflies ◽  
Emilia Romagna Region ◽  
Human Infections

Toscana virus (TOSV), transmitted by phlebotomine sandflies, is recognised as one of the most important causes of viral meningitis in summer in Mediterranean countries. A surveillance plan based on both human and entomological surveys was started in 2010 in the Emilia-Romagna region, Italy. Clinical samples from patients with neurological manifestations were collected during 2010 to 2012. The surveillance protocol was improved during these years, allowing the detection of 65 human infections. Most of these infections were recorded in hilly areas, where sandflies reach the highest density. Entomological sampling around the homes of the patients resulted in a low number of captured sandflies, while later sampling in a hilly area with high number of human cases (n=21) resulted in a larger number of captured sandflies. Using this approach, 25,653 sandflies were sampled, of which there were 21,157 females, which were sorted into 287 pools. TOSV RNA was detected by real-time PCR in 33 of the pools. The results highlighted the role of Phlebotomus perfiliewi as the main vector of TOSV and a potential link between vector density and virus circulation. This integrated system shows that an interdisciplinary approach improves the sensitiveness and effectiveness of health surveillance.

scholarly journals Development and Evaluation of Real-Time Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid and Sensitive Detection of West Nile Virus in Human Clinical Samples

2021 ◽  
Vol 10 ◽  
Author(s):  
Priyanka Singh Tomar ◽  
Sanjay Kumar ◽  
Sapan Patel ◽  
Jyoti S. Kumar
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Reverse Transcription ◽  
Febrile Illness ◽  
Posterior Uveitis ◽  
Qpcr Assay ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
West Nile Fever ◽  
West Nile

West Nile virus (WNV) causes West Nile fever and encephalitis worldwide. Currently, there are no effective drugs or vaccines available in the market to treat WNV infection in humans. Hence, it is of paramount importance to detect WNV early for the success of the disease control programs and timely clinical management in endemic areas. In the present paper, we report the development of real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for rapid and real-time detection of WNV targeting the envelope (env) gene of the virus. The RPA reaction was performed successfully at 39°C for 15 min in a real-time thermal cycler. The sensitivity of this assay was found similar to that of the quantitative real-time RT PCR (RT-qPCR) assay, which could detect 10 copies of the gene. The efficacy of the assay was evaluated with a panel of 110 WN suspected human samples showing the signs of retinitis, febrile illness and acute posterior uveitis. In comparison with RT-qPCR, RT-RPA showed a specificity of 100% (CI, 95.07–100%) and sensitivity of 96.15% (CI, 80.36–99.90%) with a negative (NPV) and positive predictive value (PPV) of 98.65 and 100%, respectively. The level of agreement between RT-RPA and reference RT-qPCR assay was shown to be very high. The turnaround time of real-time RPA assay is about 10-20 times faster than the RT-qPCR, which confirms its utility in the rapid and sensitive diagnosis of WNV infection. To the best of our knowledge, this is the first report which deals with the development of real-time RT-RPA assay for simple, rapid, sensitive, and specific detection of WNV in human clinical samples. The present RT-RPA assay proves to be a powerful tool that can be used for the rapid diagnosis of a large number of patient samples in endemic settings.

scholarly journals Detection of Viable Mycobacterium ulcerans in Clinical Samples by a Novel Combined 16S rRNA Reverse Transcriptase/IS2404 Real-Time qPCR Assay

2012 ◽  
Vol 6 (8) ◽  
pp. e1756 ◽  
Author(s):  
Marcus Beissner ◽  
Dominik Symank ◽  
Richard Odame Phillips ◽  
Yaw Ampem Amoako ◽  
Nana-Yaa Awua-Boateng ◽  
...  
Keyword(s):  
16S Rrna ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Mycobacterium Ulcerans ◽  
Qpcr Assay ◽  
Clinical Samples ◽  
Real Time Qpcr

scholarly journals Establishment of a Duplex Real-time qPCR Method for Detection of Salmonella Spp. And Serratia Fonticola in Fishmealestablishment of a Duplex Real-time qPCR Method for Detection of Salmonella Spp. And Serratia Fonticola in Fishmeal

2020 ◽  
Author(s):  
Jinghua Ruan ◽  
Wujun Wang ◽  
Tiying Zhang ◽  
Teng Zheng ◽  
Jing Zheng ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
High Specificity ◽  
Clinical Samples ◽  
Amplification Efficiency ◽  
Salmonella Spp ◽  
Specificity And Sensitivity ◽  
Qpcr Method ◽  
Real Time Qpcr

Abstract Salmonella spp. is a high-risk bacterial pathogen that is monitored in imported animal-derived feedstuffs. Serratiafonticola is the bacterial species most frequently confused with Salmonella spp. in traditional identification methods based on biochemical characteristics, which are time-consuming and labor-intensive, and thus unsuitable for daily inspection and quarantine work. In this study, we established a duplex real-time qPCR method with invA- and gyrB-specific primers and probes corresponding to Salmonella spp. and S. fonticola. The method could simultaneously detect both pathogens in imported feedstuffs, with a minimum limit of detection for Salmonella spp. and S. fonticola of 197 copies/SL and 145 copies/SL, respectively (correlation coefficient R2 = 0.999 in both cases). The amplification efficiency for Salmonella spp. and S. fonticola was 98.346% and 96.49%, respectively. Detection of clinical samples was consistent with method GB/T 13091-2018, and all seven artificially contaminated imported feed samples were positively identified. Thus, the developed duplex real-time qPCR assay displays high specificity and sensitivity, and can be used for the rapid and accurate detection of genomic DNA from Salmonella spp. and S. fonticola within hours. This represents a significant improvement in the efficiency of detection of both pathogens in imported feedstuffs.

scholarly journals Validation of Nonnested and Real-Time PCR for Diagnosis of Sheep-Associated Malignant Catarrhal Fever in Clinical Samples

2007 ◽  
Vol 19 (4) ◽  
pp. 405-408 ◽  
Author(s):  
Donald L. Traul ◽  
Naomi S. Taus ◽  
J. Lindsay Oaks ◽  
Donal O' Toole ◽  
Fred R. Rurangirwa ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
High Specificity ◽  
Clinical Samples ◽  
Malignant Catarrhal Fever ◽  
Fatal Disease ◽  
Tissue Samples ◽  
Specificity And Sensitivity ◽  
Experimentally Induced

Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.

scholarly journals Establishment and Application of a duplex real-time qPCR method for detection ofSalmonellaspp. andSerratia fonticolain imported feedstuffs

2018 ◽  
Author(s):  
Jing hua Ruan ◽  
Wu jun Wang ◽  
Quan yang Bai ◽  
Ti yin Zhang ◽  
Teng zheng ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
High Specificity ◽  
Accurate Method ◽  
Clinical Samples ◽  
Amplification Efficiency ◽  
Specificity And Sensitivity ◽  
Qpcr Method ◽  
Real Time Qpcr

AbstractSalmonellaspp. is a high-risk bacterial pathogen that is monitored in imported animal-derived feedstuffs.Serratia fonticolais the bacterial species most frequently confused withSalmonellaspp. in traditional identification methods based on biochemical characteristics, which are time-consuming and labor-intensive, and thus unsuitable for daily inspection and quarantine work. In this study, we established a duplex real-time qPCR method withinvA-andgyrB-specific primers and probes corresponding toSalmonellaspp. andS. fonticola. The method could simultaneously detect both pathogens in imported feedstuffs, with a minimum limit of detection forSalmonellaspp. andS. fonticolaof 197 copies/μL and 145 copies/μL, respectively (correlation coefficient R2= 0.999 in both cases). The amplification efficiency forSalmonellaspp. andS. fonticolawas 98.346% and 96.49%, respectively. Detection of clinical samples was consistent with method GB/T 13091-2002, and all 20 artificially contaminated imported feed samples were positively identified. Thus, the developed duplex real-time qPCR assay displays high specificity and sensitivity, and can be used for the rapid and accurate detection of genomic DNA fromSalmonellaspp. andS. fonticolawithin hours. This represents a significant improvement in the efficiency of detection of both pathogens in imported feedstuffs.ImportanceImported feedstuffs must be tested for pathogenicSalmonellaspecies that represent a biological hazard. Variousnon-Salmonellacolony-forming species belong toEnterobacteriaceae,andSerratia fonticolaforms colonies of similar color and morphology toSalmonellaspp., leading to confusion in daily quarantine tests. Traditional methods based on biochemical and serological characteristics are cumbersome and labor-intensive, and unable to fully support current quarantine testing demands. Thus, there is an urgent need to develop a rapid and accurate method for the effective identification of these pathogens. The duplex real-time qPCR method established herein can rapidly identifySalmonellaspp. andS. fonticola, and has great potential for application to feed safety and prevention of exterior pathogens.

scholarly journals Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

2015 ◽  
Vol 53 (3) ◽  
pp. 930-940 ◽  
Author(s):  
Iker A. Sevilla ◽  
Elena Molina ◽  
Natalia Elguezabal ◽  
Valentín Pérez ◽  
Joseba M. Garrido ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Reference Method ◽  
Clinical Samples ◽  
Dna Amount ◽  
Content Type ◽  
Specificity And Sensitivity

Mycobacterium tuberculosiscomplex,Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection ofMycobacteriumgenus,M. aviumsubspecies, andM. tuberculosiscomplex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n= 38) and nonmycobacterial (n= 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n= 57), archival clinical samples (n= 233), and strains isolated from various hosts (n= 147). The minimum detectable DNA amount per reaction was 50 fg forM. bovisBCG andM. kansasiiand 5 fg forM. aviumsubsp.hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identifyM. aviumandM. tuberculosiscomplex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.

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