Multiplex RT-PCR Method for the Simultaneous Detection of Nine Grapevine Viruses

Abstract The feline calicivirus (FCV), feline herspesvirus 1 (FHV-1) and feline panleukemia virus (FPV) are heavily threaten the health of cats. In this study, a triplex TaqMan real-time polymerase chain reaction (RT-PCR) assay (triplex assay) was developed to detect these viruses. The optimized concentration of primers was 0.5 µM of each, probes concentration was 0.1 µM for FCV and FHV-1, 0.05 µM for FPV. The annealing temperature was set at 54 ℃. The triplex RT-PCR assay was carefully validated. The detection limit for FPV, FCV, and FHV-1 was 5×101 copies/µL, which showed a 10-100-fold increase in the sensitivity compared with the conventional PCR. The coefficients of variation (CV) of the intra-assay variability of the test were < 1.86%, and that of inter-assay was < 3.19%, indicating excellent repeatability and reproducibility of the triplex assay. Additionally, the assay has perfect specificity. In a pilot study, samples from 48 cats were analyzed using the triplex RT-PCR method and the commercial kits, and further confirmed by sequencing. The positive rates for the samples analyzed with these two methods were 70.83% and 62.5%, respectively, which demonstrated that the developed method was more accurate than the commercial kits in clinical diagnosis.

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One-step Multiplex RT-PCR Method for Simultaneous Detection of Seed Transmissible Bacteria and Viruses in Pepper and Tomato Seeds

Research in Plant Disease ◽

10.5423/rpd.2011.17.1.044 ◽

2011 ◽

Vol 17 (1) ◽

pp. 44-51

Author(s):

Kyu-Sik Jeong ◽

Eun-Hee Soh

Keyword(s):

Simultaneous Detection ◽

Rt Pcr ◽

Tomato Seeds ◽

One Step ◽

Pcr Method

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Primary screening of blood donors by nat testing for HCV-RNA: development of an "in-house" method and results

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652007000300008 ◽

2007 ◽

Vol 49 (3) ◽

pp. 177-185 ◽

Cited By ~ 11

Author(s):

Silvano Wendel ◽

José Eduardo Levi ◽

Deise Tihe Takaoka ◽

Isabela Cristina Silva ◽

Juliana Polachini de Castro ◽

...

Keyword(s):

Blood Donors ◽

Simultaneous Detection ◽

External Control ◽

Hcv Rna ◽

Rt Pcr ◽

Primary Screening ◽

Hit Rate ◽

Whole Process ◽

Pcr Method ◽

Control Samples

An "in-house" RT-PCR method was developed that allows the simultaneous detection of the RNA of the Hepatitis C Virus (HCV) and an artificial RNA employed as an external control. Samples were analyzed in pools of 6-12 donations, each donation included in two pools, one horizontal and one vertical, permitting the immediate identification of a reactive donation, obviating the need for pool dismembering. The whole process took 6-8 hours per day and results were issued in parallel to serology. The method was shown to detect all six HCV genotypes and a sensitivity of 500 IU/mL was achieved (95% hit rate). Until July 2005, 139,678 donations were tested and 315 (0.23%) were found reactive for HCV-RNA. Except for five false-positives, all 310 presented the corresponding antibody as well, so the yield of NAT-only donations was zero, presenting a specificity of 99.83%. Detection of a window period donation, in the population studied, will probably demand testing of a larger number of donations. International experience is showing a rate of 1:200,000 - 1:500,000 of isolated HCV-RNA reactive donations.

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Simultaneous Detection and Differentiation of Chikungunya Virus and Dengue Virus in Blood at Clinical Presentation by Real Time RT-PCR

Pulse ◽

10.3329/pulse.v10i1.38605 ◽

2018 ◽

Vol 10 (1) ◽

pp. 6-11

Author(s):

MM Rahman ◽

R Rahim ◽

AH Rasel ◽

AS Murad

Keyword(s):

Dengue Virus ◽

Real Time ◽

Chikungunya Virus ◽

Clinical Presentation ◽

Viral Infections ◽

Management Strategies ◽

Simultaneous Detection ◽

Rt Pcr ◽

Show Evidence ◽

Pcr Method

Background: Chikungunya viral infection in Bangladesh has marked 2017 summer with unprecedented magnitude for the first time. Chikungunya virus (CHIKV) and Dengue virus (DENV) are arboviruses that share the same Aedes mosquito vectors and thus overlap in their endemic areas.These two viruses cause similar clinical presentations, especially in the initial stages of infection. Because the outcomes and management strategies for these two viruses are different, early and accurate diagnosis is imperative. Early diagnosis is also important for surveillance and outbreak control.Objective: As virus isolation is not undergoing in the country and antigen based commercial detection assay is not available for CHIKV we used one step real time reverse transcriptase polymerase chain reaction (RT-PCR) method to detect and discriminate CHIKV and DENV in blood during suspicious clinical symptoms.Results: By this RT-PCR method we have detected 603 cases of CHIKV and 233 cases of DENV and thus facilitated rapid diagnosis and clinical management in the recent CHIKV outbreaks in the country.Conclusion: This is the first report about molecular detection and differentiation of CHIKV and DENV at time of clinical presentation and further show evidence of simultaneous outbreaks of both the viral infections in the country.Pulse Vol.10 January-December 2017 p.6-11

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