Environmental Detection of Genogroup I, II, and IV Noroviruses by Using a Generic Real-Time Reverse Transcription-PCR Assay

ABSTRACTNorovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups.

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Investigations on the Frequency of Norovirus Contamination of Ready-to-Eat Food Items in Istanbul, Turkey, by Using Real-Time Reverse Transcription PCR

Journal of Food Protection ◽

10.4315/0362-028x.jfp-10-475 ◽

2011 ◽

Vol 74 (5) ◽

pp. 840-843 ◽

Cited By ~ 9

Author(s):

AYSUN YILMAZ ◽

KAMIL BOSTAN ◽

EDA ALTAN ◽

KARLO MURATOGLU ◽

NURI TURAN ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Epidemiological Studies ◽

Rt Pcr ◽

Pcr Assay ◽

Food Items ◽

Reverse Transcription Pcr ◽

Significant Difference ◽

Tomato Sample ◽

Pcr Assays

Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.

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Combined Immunomagnetic Separation-Molecular Beacon-Reverse Transcription-PCR Assay for Detection of Hepatitis A Virus from Environmental Samples

Applied and Environmental Microbiology ◽

10.1128/aem.70.7.4371-4374.2004 ◽

2004 ◽

Vol 70 (7) ◽

pp. 4371-4374 ◽

Cited By ~ 34

Author(s):

Khaled H. Abd El Galil ◽

M. A. El Sokkary ◽

S. M. Kheira ◽

Andre M. Salazar ◽

Marylynn V. Yates ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Environmental Samples ◽

Hepatitis A ◽

Molecular Beacon ◽

Hepatitis A Virus ◽

Immunomagnetic Separation ◽

Rt Pcr ◽

Pcr Assay ◽

The Real

ABSTRACT In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.

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Development, Evaluation, and Standardization of a Real-Time TaqMan Reverse Transcription-PCR Assay for Quantification of Hepatitis A Virus in Clinical and Shellfish Samples

Applied and Environmental Microbiology ◽

10.1128/aem.02660-05 ◽

2006 ◽

Vol 72 (6) ◽

pp. 3846-3855 ◽

Cited By ~ 313

Author(s):

M. Isabel Costafreda ◽

Albert Bosch ◽

Rosa M. Pintï¿½

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Accurate Estimation ◽

Acid Extraction ◽

Rt Pcr ◽

Pcr Assay ◽

Reverse Transcription Pcr ◽

Development Evaluation

ABSTRACT A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5′ noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness.

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Detection of a novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome in Henan Province, China, using real-time reverse transcription PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.049577-0 ◽

2013 ◽

Vol 62 (7) ◽

pp. 1060-1064 ◽

Cited By ~ 8

Author(s):

Xueyong Huang ◽

Licheng Liu ◽

Yanhua Du ◽

Hongxia Ma ◽

Yujiao Mu ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

High Sensitivity ◽

Cross Reactivity ◽

Henan Province ◽

Clinical Samples ◽

Rt Pcr ◽

Pcr Assay ◽

Serum Samples ◽

Reverse Transcription Pcr

A novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome (FTLS) was discovered in Henan Province, China. Here, we report the development of an assay for this novel bunyavirus based on real-time reverse transcription PCR (RT-PCR). The assay exhibited high sensitivity and specificity without cross-reactivity towards 13 other viruses that cause similar symptoms. To evaluate the performance of this assay in detecting clinical samples, we analysed 261 serum samples from patients in Henan Province between 2007 and 2010. Of these samples, 91.95 % were bunyavirus positive. Compared with serological assays, the real-time PCR assay was much more sensitive in identifying infected patients 1 to 7 days after the onset of symptoms.

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Evaluation of a Real-Time Reverse Transcription-PCR (RT-PCR) Assay for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Clinical Samples from an Outbreak in South Korea in 2015

Journal of Clinical Microbiology ◽

10.1128/jcm.00667-17 ◽

2017 ◽

Vol 55 (8) ◽

pp. 2554-2555 ◽

Cited By ~ 5

Author(s):

Jee-Soo Lee ◽

Ji Soo Ahn ◽

Byeong Su Yu ◽

Sung Im Cho ◽

Man Jin Kim ◽

...

Keyword(s):

Middle East ◽

South Korea ◽

Real Time ◽

Reverse Transcription ◽

Middle East Respiratory Syndrome ◽

Clinical Samples ◽

Rt Pcr ◽

Pcr Assay ◽

Reverse Transcription Pcr

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Differentiation of PEDV Classical Attenuated Vaccine Strains from Wild-type Strains using One-Step Real-Time Fluorescent Reverse Transcription PCR Assay Targeting ORF1 Nucleotides Deletion Region

10.21203/rs.3.rs-529609/v1 ◽

2021 ◽

Author(s):

Zhilin Wang ◽

Xuerui Li ◽

Youjun Shang ◽

Jinyan Wu ◽

Zhen Dong ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Economic Losses ◽

Wild Type ◽

Rt Pcr ◽

Pcr Assay ◽

Attenuated Vaccine ◽

Reverse Transcription Pcr ◽

One Step ◽

Type Strains

Abstract BackgroundPorcine epidemic diarrhea virus (PEDV) is a pathogen causing serious disease and resulting in severe economic losses in the swine industry. In recent years, although China has adopted a large-scale vaccine immunization strategy, many types of PEDV strains, including classical attenuated vaccine strains, have been discovered in the immunized pig herds. Therefore, monitoring the prevalence of different types of PEDV strains is particularly important for the production of pigs and the safety evaluation of related attenuated vaccines MethodsIn the study, a one-step real-time fluorescent reverse transcription PCR (one-step real-time RT-PCR) assay targeting 24-nucleotide deletion in the ORF1 region of three PEDV classical attenuated vaccine strains (derived from classical strains) was established, which could effectively distinguish PEDV classical attenuated vaccine strains and wild-type strains. ResultsIn our study, the RNA detection limits for PEDV wild-type strains and classical attenuated vaccine strains were 3.0×103 copies and 3.0×102 copies, respectively. This assay was highly specific for PEDV, with no cross-reactivity for other viruses, causing diarrheal disease. A total of 117 swine fecal samples were analysed by this established real-time RT-PCR assay, indicating that classical attenuated vaccine strains were present in the swine herds in Gansu province, China. Additionally, a pair of primers and two probes of the established assay can be placed in one reaction tube to distinguish PEDV classical attenuated vaccine strains and wild-type strains. ConclusionOur results provided an effective and cheap technology platform for clinical rapid identification testing and epidemiological investigations of PEDV wild-type strains and classical attenuated vaccine strains

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Implementation of an in-house real-time reverse transcription-PCR assay to detect the emerging SARS-CoV-2 N501Y variants

Journal of Clinical Virology ◽

10.1016/j.jcv.2021.104868 ◽

2021 ◽

pp. 104868

Author(s):

Marielle BEDOTTO ◽

Pierre-Edouard FOURNIER ◽

Linda HOUHAMDI ◽

Philippe COLSON ◽

Didier RAOULT

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Pcr Assay ◽

Reverse Transcription Pcr

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Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus

Journal of Virological Methods ◽

10.1016/j.jviromet.2006.09.022 ◽

2007 ◽

Vol 139 (2) ◽

pp. 150-158 ◽

Cited By ~ 17

Author(s):

O. Guionie ◽

D. Toquin ◽

E. Sellal ◽

S. Bouley ◽

F. Zwingelstein ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Laboratory Evaluation ◽

Pcr Assay ◽

Avian Metapneumovirus ◽

Reverse Transcription Pcr ◽

Detection And Identification

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Comet and cup genes in Drosophila spermatogenesis: the first demonstration of post-meiotic transcription

Biochemical Society Transactions ◽

10.1042/bst0360540 ◽

2008 ◽

Vol 36 (3) ◽

pp. 540-542 ◽

Cited By ~ 11

Author(s):

Carine Barreau ◽

Elizabeth Benson ◽

Helen White-Cooper

Keyword(s):

In Situ Hybridization ◽

Expression Pattern ◽

Reverse Transcription ◽

Protein Product ◽

Rt Pcr ◽

Pcr Assay ◽

Single Cyst ◽

Reverse Transcription Pcr

Post-meiotic transcription is widespread in mammalian spermatogenesis, but is generally believed to be absent from Drosophila spermatogenesis. Genes required during meiosis, in early spermatids or later in spermiogenesis are typically transcribed in primary spermatocytes in Drosophila. Their mRNAs are then stored in the cytoplasm until the protein product is needed. Recently, using in situ hybridization, we identified 17 Drosophila genes, collectively named ‘comets’ and ‘cups’, whose mRNAs are most abundant in, and localize to the distal ends of, elongating spermatids. Using a single-cyst quantitative RT–PCR (reverse transcription–PCR) assay, we confirmed this unusual expression pattern and conclusively demonstrate the existence of post-meiotic transcription in Drosophila spermatids. We found that transcription of comets and cups occurs just before protamines can be detected in spermatid nuclei.

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