Comparative evaluation of RT-PCR, nucleic acid sequence-based amplification (NASBA) and real-time RT-PCR for detection of noroviruses in faecal material

2006 ◽  
Vol 135 (2) ◽  
pp. 163-172 ◽  
Author(s):  
Alain Houde ◽  
Danielle Leblanc ◽  
Elyse Poitras ◽  
Pierre Ward ◽  
Julie Brassard ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Comparative Evaluation ◽  
Nucleic Acid Sequence ◽  
Rt Pcr ◽  
Faecal Material

scholarly journals Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay

2006 ◽  
Vol 72 (8) ◽  
pp. 5349-5358 ◽  
Author(s):  
Saskia A. Rutjes ◽  
Harold H. J. L. van den Berg ◽  
Willemijn J. Lodder ◽  
Ana Maria de Roda Husman
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Water Samples ◽  
Rna Extraction ◽  
Sewage Treatment ◽  
Rdrp Gene ◽  
Nucleic Acid Sequence ◽  
Viral Gastroenteritis ◽  
Rt Pcr ◽  
Inhibitory Factors

ABSTRACT Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment.

scholarly journals Evaluation of real-time nucleic acid sequence-based amplification for detection of Chikungunya virus in clinical samples

2009 ◽  
Vol 58 (9) ◽  
pp. 1168-1172 ◽  
Author(s):  
J.-N. Telles ◽  
K. Le Roux ◽  
P. Grivard ◽  
G. Vernet ◽  
A. Michault
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Chikungunya Virus ◽  
False Negative ◽  
Nucleic Acid Sequence ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Plasma Samples ◽  
Rt Pcr ◽  
Nucleic Acid Extraction

The Chikungunya virus (CHIKV) is a member of the genus Alphavirus that is transmitted to humans by Aedes mosquitoes. In 2005 and 2006, the Indian Ocean island of La Réunion was hit with an unprecedented CHIKV fever outbreak that infected 300 000 people. In the present study, we describe the evaluation of real-time nucleic acid sequence-based amplification (RT-NASBA) for the detection of CHIKV in clinical samples. A co-extracted and co-amplified chimerical CHIKV RNA sequence was used as an internal control to eliminate false-negative results. The detection threshold of the assay was determined from quantified CHIKV-positive plasma, and estimated to be 200 copies per NASBA reaction. The specificity of the assay was determined using blast analyses and non-cross-reactivity using an O'nyong-nyong virus culture and 250 CHIKV RT-PCR-negative plasma samples. A 100 % specificity was found and no invalid result was obtained, showing the good quality of the nucleic acid extraction. The assay was then evaluated using 252 CHIKV-positive RT-PCR plasma samples. The samples were all tested positive, including those with low viral load. This evaluation showed that the RT-NASBA is a rapid (5 h from sample nucleic acid extraction to detection), sensitive, specific and reliable method for the routine diagnosis of CHIKV in clinical samples.

Comparative detection of rotavirus RNA by conventional RT-PCR, TaqMan RT-PCR and real-time nucleic acid sequence-based amplification

2015 ◽  
Vol 213 ◽  
pp. 1-4 ◽  
Author(s):  
Qiu-Hua Mo ◽  
Hai-Bo Wang ◽  
Hua Tan ◽  
Bi-Mei Wu ◽  
Zi-Li Feng ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Nucleic Acid Sequence ◽  
Rt Pcr

Rapid and simultaneous detection of three major diarrhea-causing viruses by multiplex real-time nucleic acid sequence-based amplification

2015 ◽  
Vol 160 (3) ◽  
pp. 719-725 ◽  
Author(s):  
Qiu-Hua Mo ◽  
Hai-Bo Wang ◽  
Hui-Rong Dai ◽  
Ji-Can Lin ◽  
Hua Tan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Simultaneous Detection ◽  
Nucleic Acid Sequence

scholarly journals Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A

2009 ◽  
Vol 39 (5) ◽  
pp. 1445-1451 ◽  
Author(s):  
Helena Lage Ferreira ◽  
Fernando Rosado Spilki ◽  
Márcia Mercês Aparecida Bianchi dos Santos ◽  
Renata Servan de Almeida ◽  
Clarice Weis Arns
Keyword(s):  
Real Time ◽  
Comparative Evaluation ◽  
Virus Isolation ◽  
Diagnostic Methods ◽  
N Gene ◽  
Genomic Rna ◽  
Rt Pcr ◽  
Avian Metapneumovirus ◽  
Lower Detection ◽  
Subtype A

Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an established test for the attachment (G) gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F- based RRT-PCR and F-based conventional RT-PCR (10(0.3) to 10¹ TCID50 mL-1). The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N- and F-based RRT-PCR and they can be successfully used for AMPV/A detection.

scholarly journals Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

2005 ◽  
Vol 71 (11) ◽  
pp. 7113-7116 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Nucleic Acid Sequence ◽  
The Real ◽  
Nasba Assay ◽  
Environmental Pathogens

ABSTRACT A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA 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, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.

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