Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods

Jean, Julie; D’Souza, Doris H.; Jaykus, Lee Ann

doi:10.1128/aem.70.11.6603-6610.2004

Cited by 66 publications

(43 citation statements)

References 28 publications

(28 reference statements)

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“…All of these studies used PCR amplification of a single targeted gene segment of a given bacterium with a single set of primers. In the m-PCR, where several sets of primers are used in the reaction for amplification of multiple target genes, the diagnostic sensitivity or detection limit is generally considered to be low (Rosenfield & Jaykus 1999, Jean et al 2004, Lee et al 2005). In at least 1 study, a 10-fold decrease in detection sensitivity compared to single PCR was recorded (Jean et al 2004).…”

Section: Discussionmentioning

confidence: 99%

“…In the m-PCR, where several sets of primers are used in the reaction for amplification of multiple target genes, the diagnostic sensitivity or detection limit is generally considered to be low (Rosenfield & Jaykus 1999, Jean et al 2004, Lee et al 2005). In at least 1 study, a 10-fold decrease in detection sensitivity compared to single PCR was recorded (Jean et al 2004). In an m-PCR-based method for simultaneous detection of 4 pathogens involved in warm-water streptococcosis in fish tissues, Mata et al (2004) recorded detection limits of 5 × 10 3 cells g -1 of tissue for Streptococcus iniae, 1.2 × 10 4 cells g -1 for S. difficilis, 1 × 10 4 cells g -1 for S. paraubiris, and 2.5 × 10 3 cells g -1 for Lactococcus garviae.…”

Section: Discussionmentioning

confidence: 99%

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Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila

Panangala

Shoemaker²,

Santen³

et al. 2007

Dis. Aquat. Org.

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A multiplex PCR (m-PCR) method was developed for simultaneous detection of 3 important fish pathogens in warm water aquaculture. The m-PCR to amplify target DNA fragments from Flavobacterium columnare (504 bp), Edwardsiella ictaluri (407 bp) and Aeromonas hydrophila (209 bp) was optimized by adjustment of reaction buffers and a touchdown protocol. The lower detection limit for each of the 3 bacteria was 20 pg of nucleic acid template from each bacteria per m-PCR reaction mixture. The sensitivity threshold for detection of the 3 bacteria in tissues ranged between 3.4 × 10 2 and 2.5 × 10 5 cells g -1 of tissue (channel catfish Ictalurus punctatus Rafinesque). The diagnostic sensitivity and specificity of the m-PCR was evaluated with 10 representative isolates of each of the 3 bacteria and 11 other Gram-negative and 2 Gram-positive bacteria that are taxonomically related or ubiquitous in the aquatic environment. Except for a single species (A. salmonicida subsp. salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. m-PCR was compared with bacteriological culture for identification of bacteria in experimentally infected fish. The m-PCR appears promising for the rapid, sensitive and simultaneous detection of Flavobacterium columnare, E. ictaluri and A. hydrophila in infected fish compared to the timeconsuming traditional bacteriological culture techniques. KEY WORDS: Multiplex-PCR · Fish · Edwardsiella · Flavobacterium · AeromonasResale or republication not permitted without written consent of the publisher Dis Aquat Org 74: 199-208, 2007 most important bacterial diseases affecting the aquaculture industry in the USA (United States Department of Agriculture 2003). The prevalence and impact of septicemic disease in fish caused by Aeromonas hydrophila (referred to as motile aeromonad septicemia, MSA), is less known. However, A. hydrophila is ubiquitous in aquatic ecosystems (Holmes et al. 1996) and recent studies have shown that catfish latently infected with A. hydrophila (carriers) could shed the organism when co-infected with Edwardsiella ictaluri (Nusbaum & Morrison 2002). Since all 3 species of bacteria (Flavobacterium columnare, E. ictaluri, and A. hydrophila) are ubiquitous in the aquatic environment and fish are reared in extensive pond acreages with high stocking densities (20 000 to 30 000 fish ha -1 ), the occurrence of coinfections or multiple infections in the same host should not be overlooked (Jack et al. 1992). Traditional methods of diagnosing bacterial infections using culture techniques require several days to arrive at a definitive diagnosis, resulting in delayed implementation of control measures and increased potential for spreading of disease. Because PCR can target unique genetic sequences of microorganisms, PCR-based nucleic acid amplification techniques have gained recognition as rapid, sensitive, and specific methods for detection of disease-causing pathogens in various aquaculture species (Vantarakis et al. 2000, Del Cero e...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila

Panangala

Shoemaker²,

Santen³

et al. 2007

Dis. Aquat. Org.

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“…Using established primer pairs, multiplex NASBA assays were developed for simultaneous detection of HAV and NoV GI and GII in spiked ready-to-eat foods. All three viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) RT-PCR units [91].…”

Section: Nucleic Acid Sequence-based Amplification (Nasba)mentioning

confidence: 88%

Nucleic Acid Detection of Major Foodborne Viral Pathogens Human: Noroviruses and Hepatitis A Virus

Chen¹

2016

Nucleic Acids - From Basic Aspects to Laboratory Tools

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Human noroviruses (hNoVs) and hepatitis A virus (HAV) are the leading cause of foodborne viral illness, and they exact a considerable economic and health toll worldwide. The detection of viral contamination in foods requires highly sensitive and accurate methods, due to the intrinsically low amounts of viruses and the complexity of the sample matrices. In recent years, a wide variety of nucleic acid-based molecular methods have been developed for the detection of hNoVs and HAV, displaying superior sensitivity, specificity, and speed. This chapter aims to provide a summary of the application of the molecular methods for the detection of the two important foodborne viruses.

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“…There are several reports on multiplex detection of microorganisms using NASBA. In a research conducted by Jean et al (22) multiplex NASBA had been used for simultaneous detection of intestinal viruses which cause food poisoning. In another investigation, Lau et al (11) used this method for the detection of respiratory system infectious viruses.…”

Section: Discussionmentioning

confidence: 99%

A Simple and Rapid Method for the Detection of HIV-1/HCV in Co-Infected Patients

et al. 2013

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Background: Due to some limitations of serological methods in diagnosis of patients infected with HIV-1 (human immunodeficiency virus) and HCV (hepatitis C virus), it is profoundly important to use molecular methods for the detecting of these infectious agents. However, the most significant problems are the exorbitant cost of these methods and the need of a thermocycler which is an expensive instrument. Objectives: The current research recruits a multiplex nucleic acid sequence base amplification (NASBA) in order to simultaneously detect HIV-1 and HCV genomes in patients' plasma samples. Sensitivity and specificity of this method have been evaluated using clinical samples. Materials and Methods: A multiplex NASBA assay for simultaneous detection of HCV and HIV-1 by the use of specific primers were designed and validated. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used. A total of 40 samples of HIV-1 (20 samples) and HCV (20 samples) were used in the NASBA assay. The specificity and sensitivity of the assay were evaluated. Results: Our results have demonstrated that the primers used in the assay had no interrelation with each other and other possible interfering agents in the assay. The analytical sensitivity of the assay for both HIV-1 and HCV was determined to be 1000 copies/mL and the clinical sensitivity and specificity were 93.3% and 100%, respectively. Conclusions: By exploiting this multiplex NASBA assay, it is possible to detect HIV-1 and HCV infection/co-infection in patients' plasma with a suitable sensitivity and specificity. Furthermore, due to its simplicity and multiplexing feature, it could be used in limited access laboratories in a cost-effective manner.

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Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods

Cited by 66 publications

References 28 publications

Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila

Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila

Nucleic Acid Detection of Major Foodborne Viral Pathogens Human: Noroviruses and Hepatitis A Virus

A Simple and Rapid Method for the Detection of HIV-1/HCV in Co-Infected Patients

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