scholarly journals Norovirus Detection in Ready-To-Eat Salads by Propidium Monoazide Real Time RT-PCR Assay

2020 ◽  
Vol 10 (15) ◽  
pp. 5176
Author(s):  
Valentina Terio ◽  
Patrizio Lorusso ◽  
Anna Mottola ◽  
Canio Buonavoglia ◽  
Giuseppina Tantillo ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Detection ◽  
Virus Infectivity ◽  
Propidium Monoazide ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Genotype I ◽  
Food Borne ◽  
Processing Plants ◽  
Norovirus Gii

Ready-to-eat (RTE) salads have recently been associated with food-borne norovirus outbreaks, although these infections are mainly related to shellfish and berry consumption in the EU. A total of 135 bagged RTE vegetables were analyzed in order to investigate the occurrence of norovirus (NoV) genotype I (GI) and II (GII) RNA and to differentiate between infectious and non-infectious viruses by using propidium monoazide (PMAxx) coupled with the real time Reverse Transcription (RT) PCR method. Initially, the PMAxx real time RT-PCR assay was optimized on NoV GI and GII suspensions, and proved capable of detecting significant (p < 0.05) differences between infectious and inactivated viruses. Our analysis conducted on RTE salads samples showed the presence of norovirus GII in 74.8% of samples, of which 37.6% were infectious. The samples tested for viral contamination came from only two RTE vegetable-processing plants. The findings in this study could also be due to virally-contaminated water used in food production, processing, or preparation. This study stresses the need for effective real-time RT-PCR tools capable of qualitative and quantitative detection of NoV RNA, as well as being able to measure virus infectivity, for risk assessment, which is crucial in several public health measures and food regulations.

Real-time PCR assay for sensitive organ detection and epidemic investigation of Turbot reddish body iridovirus

2009 ◽  
Vol 6 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Wu Cheng-Long ◽  
Shi Cheng-Yin ◽  
Huang Jie ◽  
Kong Xiao-Yu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Viral Genome ◽  
Sybr Green ◽  
Sybr Green I ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Linear Coefficient ◽  
Sensitive Organ

AbstractA rapid and sensitive real-time polymerase chain reaction (PCR) assay coupled with SYBR Green I chemistry was developed for the quantitative detection of Turbot reddish body iridovirus (TRBIV) isolated from farmed turbot (Scophthalmus maximus). A 152 bp DNA fragment from the TRBIV major capsid protein (MCP) gene was involved in the real-time PCR (RT-PCR) assay using the Roter Gene 3000 sequence detection system. The PCR mixture contained a fluorescent dye, SYBR Green I, which exhibited fluorescence enhancement when bound to double-stranded (ds) DNA. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. The positive control plasmid, pUCm-T/TRBIV MCP, containing the target sequence, was quantified to make a standard curve for sample detection after serial tenfold dilution. Linear coefficient correlations between the cycle threshold (CT) value and logarithmic positive plasmid concentration were close to one (R2=0.9952) and the detection limit of the assay was 102 copies of positive plasmids. The quantitative detection of virus in different tissues from TRBIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (5.23×106 and 2.18×106 viral genome copies/mg tissue, respectively), while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed that many cultured turbots were infected and TRBIV has become epidemic in turbot farms located along the Shandong peninsula. The virus number varied from 1.27×102 to 2.33×106 viral genome copies/mg tissue in spleens of infected turbot. These results suggest that the RT-PCR assay reported here can be used as a rapid, sensitive and quantitative method for TRBIV.

scholarly journals A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

2005 ◽  
Vol 71 (12) ◽  
pp. 9008-9012 ◽  
Author(s):  
David Rodríguez-Lázaro ◽  
Maria Pla ◽  
Mariela Scortti ◽  
Héctor J. Monzó ◽  
José A. Vázquez-Boland
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Internal Amplification Control ◽  
Taqman Probe ◽  
Quantitative Detection ◽  
Pcr Assay ◽  
Double Stranded Dna ◽  
Specificity And Sensitivity ◽  
Food Borne ◽  
Smoked Salmon

ABSTRACT We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R 2) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.

scholarly journals Development and application of a TaqMan-MGB real-time RT-PCR assay for the detection of porcine epidemic diarrhoea virus strains in China

2016 ◽  
Vol 60 (2) ◽  
pp. 127-133 ◽  
Author(s):  
Yi-Xuan Hou ◽  
Chun Xie ◽  
Kang Wang ◽  
Yu-Ting Zhao ◽  
Yang-Yang Xie ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
N Gene ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Conserved Sequence ◽  
Porcine Epidemic Diarrhoea Virus ◽  
Diarrhoea Virus ◽  
Quantitative Pcr Assay

AbstractIntroduction:A real-time RT-PCR method for identification and quantification of porcine epidemic diarrhoea virus (PEDV) strains in China was developed.Material and Methods:Based on the conserved sequence of the PEDV nucleocapsid (N) gene, a primer pair and probe were designed to establish a TaqMan-MGB real-time RT-PCR assay for quantitative detection of the virus. The sequence was cloned into the pMD18-T vector and a series of diluted recombinant plasmids were used to generate a standard curve with an R2 value of 0.999.Results:The developed quantitative PCR assay detected viral titres as low as 0.1 TCID50with high specificity and no cross-reaction with other porcine viruses (PoRV, TGEV, PRRSV, or CSFV). The intra-batch and inter-batch coefficients of variation were both less than 1%, which indicated good reproducibility. Thirty clinical diarrhoea samples obtained from pigs in Shanghai and Fujian were analysed using this quantitative PCR assay. Out of these samples, 93.3% were found to be PEDV positive.Conclusion:This approach is suitable for clinical sample identification and pathogenesis studies.

Real time RT-PCR assay for quantitative detection ofCitrus viroid IIIin plant tissues

2009 ◽  
Vol 58 (1) ◽  
pp. 181-185 ◽  
Author(s):  
S. Rizza ◽  
G. Nobile ◽  
M. Tessitori ◽  
A. Catara ◽  
E. Conte
Keyword(s):  
Real Time ◽  
Quantitative Detection ◽  
Plant Tissues ◽  
Rt Pcr ◽  
Pcr Assay

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

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Siti Tasnim Makhtar ◽  
Sheau Wei Tan ◽  
Nur Amalina Nasruddin ◽  
Nor Azlina Abdul Aziz ◽  
Abdul Rahman Omar ◽  
...  
Keyword(s):  
Real Time ◽  
Measles Virus ◽  
Clinical Sample ◽  
N Gene ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
High Morbidity ◽  
Canine Distemper ◽  
Rt Pcr ◽  
Pcr Assay

Abstract Background Morbilliviruses 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. Results A 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. Conclusions The 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.

scholarly journals Development of Taqman-Based Real-Time RT-PCR Assay Based on N Gene for The Quantitative Detection of Feline Morbillivirus

2020 ◽  
Author(s):  
Siti Tasnim Makhtar ◽  
Sheau Wei Tan ◽  
Nur Amalina Nasruddin ◽  
Nor Azlina Abdul Aziz ◽  
Abdul Rahman Omar ◽  
...  
Keyword(s):  
Real Time ◽  
Measles Virus ◽  
Clinical Sample ◽  
Feline Leukemia Virus ◽  
N Gene ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Canine Distemper ◽  
Rt Pcr ◽  
Pcr Assay

Abstract Background: Feline morbillivirus (FeMV) is a member of genus Morbillivirus which has been associated with the chronic kidney disease in cats even though a definite relationship is still unclear. Morbilliviruses are associated with severe diseases such as Peste des petits ruminants, canine distemper and measles. FeMV has been detected in many countries including Malaysia. 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.Results: A specific assay was developed since no amplification was observed in viral strains from the same Paramyxoviridae family, 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 x 104 copies/L 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. Besides that, the clinical sample evaluation using this assay showed a higher detection rate, with 26 (37%) 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.Conclusions: The Taqman-based real-time RT PCR assay targeting the N gene described here is more sensitive, specific, rapid and reproducible compared to the conventional RT-PCR assay targeting the N gene and it could be used to detect early infection in cats.

scholarly journals Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses

2021 ◽  
pp. 104063872199481
Author(s):  
Yixin Xiao ◽  
Fan Yang ◽  
Fumin Liu ◽  
Hangping Yao ◽  
Nanping Wu ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Real Time ◽  
Influenza A ◽  
Minor Groove Binder ◽  
Rt Pcr ◽  
Influenza A Viruses ◽  
Pcr Assay ◽  
Infectious Dose ◽  
The Matrix ◽  
Taqman Minor Groove Binder

The H2 subtypes of avian influenza A viruses (avian IAVs) have been circulating in poultry, and they have the potential to infect humans. Therefore, establishing a method to quickly detect this subtype is pivotal. We developed a TaqMan minor groove binder real-time RT-PCR assay that involved probes and primers based on conserved sequences of the matrix and hemagglutinin genes. The detection limit of this assay was as low as one 50% egg infectious dose (EID50)/mL per reaction. This assay is specific, sensitive, and rapid for detecting avian IAV H2 subtypes.

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

2013 ◽  
Vol 189 (2) ◽  
pp. 277-282 ◽  
Author(s):  
Yong Yan ◽  
Heng-hui Wang ◽  
Lei Gao ◽  
Ji-mei Ji ◽  
Zhi-jie Ge ◽  
...  
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Human Norovirus ◽  
One Step

Multicenter evaluation of the ENTEROVIRUS R-gene™ real-time RT-PCR assay for the detection of enteroviruses in clinical specimens

2010 ◽  
Vol 47 (1) ◽  
pp. 54-59 ◽  
Author(s):  
Sylvie Pillet ◽  
Geneviève Billaud ◽  
Shabir Omar ◽  
Bruno Lina ◽  
Bruno Pozzetto ◽  
...  
Keyword(s):  
Real Time ◽  
R Gene ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Clinical Specimens
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