Application of Multiplex TaqMan Real-Time PCR Assay in Survey of Five Lily Viruses Infecting Lilium spp.

Lily symptomless virus (LSV), Lily mottle virus (LMoV), Cucumber mosaic virus (CMV), Shallot yellow stripe virus (SYSV), and Plantago asiatica mosaic virus (PlAMV) are five of the economically important viruses infecting lilies (Lilium spp.) worldwide. In order to prevent the occurrence and spread of these viruses, it is necessary to develop a rapid, effective, and sensitive detection method for the simultaneous detection and specific quantification of these viruses. In this study, specific primers and probes for multiplex TaqMan real-time PCR assays designed from conserved regions of the coat protein sequence of each virus were used for the simultaneous detection of these viruses in lilies (Lilium spp.). The optimal concentration of primers and probes and reaction annealing temperature were 20 µM and 55.9 °C, respectively. The detection limits of the assay were 1.33 × 102, 1.27 × 101, 1.28 × 101, 2.33 × 102, and 2.01 × 102 copies·μL−1 for LSV, LMoV, CMV, SYSV, and PlAMV, respectively. Specificity was determined using seven viral pathogens of lilies. Variability tests of intra- and inter-assays showed high reproducibility with coefficients of variation <2%. The multiplex TaqMan real-time PCR assay was used to detect these viruses from lily samples in China. In brief, our developed assay showed high specificity, sensitivity, and reproducibility for the simultaneous detection and differentiation of five lily-infecting viruses and can be used for certification and quarantine programs.

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Quantification and Differentiation of Campylobacter jejuni and Campylobacter coli in Raw Chicken Meats Using a Real-Time PCR Method

Journal of Food Protection ◽

10.4315/0362-028x-70.9.2015 ◽

2007 ◽

Vol 70 (9) ◽

pp. 2015-2022 ◽

Cited By ~ 37

Author(s):

JOONBAE HONG ◽

WOO KYUNG JUNG ◽

JUN MAN KIM ◽

SO HYUN KIM ◽

HYE CHEONG KOO ◽

...

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Real Time Pcr ◽

Simultaneous Detection ◽

High Specificity ◽

Campylobacter Coli ◽

Pcr Assay ◽

The Real ◽

Real Time Quantitative Pcr ◽

Campylobacter Species

Campylobacter species are one of the most common causes of bacterial diarrhea in humans worldwide. The consumption of foods contaminated with two Campylobacter species, C. jejuni and C. coli, is usually associated with most of the infections in humans. In this study, a rapid, reliable, and sensitive multiplex real-time quantitative PCR was developed for the simultaneous detection, identification, and quantification of C. jejuni and C. coli. In addition, the developed method was applied to the 50 samples of raw chicken meat collected from retail stores in Korea. C. jejuni and C. coli were detected in 88 and 86% of the samples by real-time quantitative PCR and the conventional microbiological method, respectively. The specificity of the primer and probe sets was confirmed with 30 C. jejuni, 20 C. coli, and 35 strains of other microbial species. C. jejuni and C. coli could be detected with high specificity in less than 4 h, with a detection limit of 1 log CFU/ml by the developed real-time PCR. The average counts (log CFU per milliliter) of C. jejuni or C. coli obtained by the conventional methods and by the real-time PCR assay were statistically correlated with a correlation coefficient (R2) between 0.73 and 0.78. The real-time PCR assay developed in this study is useful for screening for the presence and simultaneous differential quantification of C. jejuni and C. coli.

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Progress Towards a Real-time PCR Assay for the Simultaneous Detection of Clavibacter michiganensis subsp. michiganensis and Pepino mosaic virus in Tomato Seed

Journal of Phytopathology ◽

10.1111/j.1439-0434.2012.01911.x ◽

2012 ◽

Vol 160 (7-8) ◽

pp. 353-363 ◽

Cited By ~ 6

Author(s):

Kameka L. Johnson ◽

Ron R. Walcott

Keyword(s):

Mosaic Virus ◽

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Clavibacter Michiganensis ◽

Pcr Assay ◽

Tomato Seed ◽

Pepino Mosaic Virus ◽

Clavibacter Michiganensis Subsp

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Development of a multiplex real-time PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A

Journal of Virological Methods ◽

10.1016/j.jviromet.2007.03.019 ◽

2007 ◽

Vol 144 (1-2) ◽

pp. 57-64 ◽

Cited By ~ 40

Author(s):

Wee Theng Ong ◽

Abdul Rahman Omar ◽

Aini Ideris ◽

Sharifah Syed Hassan

Keyword(s):

Influenza Virus ◽

Real Time ◽

Virus Type ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Type A ◽

Sybr Green ◽

Pcr Assay ◽

H9n2 Influenza Virus

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Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcastle disease virus, and duck Tembusu virus

BMC Veterinary Research ◽

10.1186/s12917-020-02399-z ◽

2020 ◽

Vol 16 (1) ◽

Author(s):

Xiyu Zhang ◽

Ming Yao ◽

Zhihui Tang ◽

Daning Xu ◽

Yan Luo ◽

...

Keyword(s):

Influenza Virus ◽

Standard Deviation ◽

Real Time ◽

Real Time Pcr ◽

Virus Isolation ◽

Simultaneous Detection ◽

Conventional Pcr ◽

Pcr Assay ◽

Duck Tembusu Virus ◽

Relative Standard

Abstract Background Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks. Results The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 101 copies/μL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won’t cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%. Conclusion The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks.

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A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

Journal of Clinical Microbiology ◽

10.1128/jcm.01986-20 ◽

2020 ◽

Vol 59 (1) ◽

pp. e01986-20

Author(s):

Ibne Karim M. Ali ◽

Shantanu Roy

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Small Subunit ◽

Clinical Samples ◽

Pcr Assay ◽

Content Type ◽

Rdna Sequences ◽

Appropriate Treatment

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

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Developing a multiplex real-time PCR with a new pre-enrichment to simultaneously detect four foodborne bacteria in milk

Future Microbiology ◽

10.2217/fmb-2019-0044 ◽

2019 ◽

Vol 14 (10) ◽

pp. 885-898 ◽

Cited By ~ 2

Author(s):

Moezi Parichehr ◽

Kargar Mohammad ◽

Doosti Abbas ◽

Khoshneviszadeh Mehdi

Keyword(s):

Real Time ◽

Foodborne Pathogens ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Pcr Assay ◽

Reliable Technique ◽

E Coli ◽

Foodborne Bacteria ◽

Multiple Pathogens ◽

Enrichment Step

Aim: The aim of this study is to formulate a new single nonselective pre-enrichment medium (ELSS) that can support the concurrent growth of four major foodborne pathogens containing E. coli O157: H7, L. monocytogenes, S. aureus and S. enterica serovar Entertidis to develop a multiplex TaqMan Real-time PCR (mRT-PCR). Methods: The mRT-PCR with a new pre-enrichment was carried out for simultaneous detection and quantification of these foodborne bacteria. Results: By using mRT-PCR after 16 h pre-enrichment in ELSS, the detection limit of each pathogen was 1 CFU/25 ml contaminated milk, as well as inclusivity and exclusivity reached 100%. Conclusion: The mRT-PCR assay with pre-enrichment step is a fast and reliable technique for detecting single or multiple pathogens in food products.

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