A novel quantitative multiplex real-time RT-PCR for the simultaneous detection and differentiation of West Nile virus lineages 1 and 2, and of Usutu virus

2013 ◽  
Vol 189 (2) ◽  
pp. 321-327 ◽  
Author(s):  
Javier Del Amo ◽  
Elena Sotelo ◽  
Jovita Fernández-Pinero ◽  
Carmina Gallardo ◽  
Francisco Llorente ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
West Nile ◽  
Usutu Virus

scholarly journals Screening of Mosquitoes for West Nile Virus and Usutu Virus in Croatia, 2015–2020

2021 ◽  
Vol 6 (2) ◽  
pp. 45
Author(s):  
Ana Klobucar ◽  
Vladimir Savic ◽  
Marcela Curman Posavec ◽  
Suncica Petrinic ◽  
Urska Kuhar ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Culex Pipiens ◽  
Rt Pcr ◽  
West Nile ◽  
Usutu Virus ◽  
Culex Pipiens Complex ◽  
Mammalian Origin ◽  
Mosquito Pool ◽  
The City ◽  
Generation Sequencing

In the period from 2015 to 2020, an entomological survey for the presence of West Nile virus (WNV) and Usutu virus (USUV) in mosquitoes was performed in northwestern Croatia. A total of 20,363 mosquitoes were sampled in the City of Zagreb and Međimurje county, grouped in 899 pools and tested by real-time RT-PCR for WNV and USUV RNA. All pools were negative for WNV while one pool each from 2016 (Aedes albopictus), 2017 (Culex pipiens complex), 2018 (Cx. pipiens complex), and 2019 (Cx. pipiens complex), respectively, was positive for USUV. The 2018 and 2019 positive pools shared 99.31% nucleotide homology within the USUV NS5 gene and both clustered within USUV Europe 2 lineage. The next-generation sequencing of one mosquito pool (Cx. pipiens complex) collected in 2018 in Zagreb confirmed the presence of USUV and revealed several dsDNA and ssRNA viruses of insect, bacterial and mammalian origin.

Establishment and evaluation of real-time PCR for West Nile virus detection

2009 ◽  
Vol 6 (1) ◽  
pp. 55-59 ◽  
Author(s):  
Shi Li-Jun ◽  
Lu Mao-Min ◽  
Li Gang ◽  
Li Cheng-Yao ◽  
Zhang Jin-Gang
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Virus Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
West Nile ◽  
Capsid Protein Gene ◽  
Specific Test ◽  
Intercalating Dye ◽  
Polymerase Chain

AbstractA rapid real-time polymerase chain reaction (RT-PCR) for detecting West Nile virus (WNV) was established. Primers were designed according to the sequence of the capsid protein gene of WNV by Primer Premier 5.0. In this way, an inexpensive assay using the intercalating dye SYBR Green I was developed and validated. The amplifying curve showed that this method could successfully amplify 102 copies/μl of the WNV gene, while reference to Japanese encephalitis virus (JEV) and blank control were all negative. Tenfold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay system showed high reproducibility with coefficient of variation (CV) <2%. Thus the newly established RT-PCR assay was shown to be a rapid, sensitive and specific test for detecting WNV.

scholarly journals Development of a Real-Time Loop-Mediated Isothermal Amplification Method for the Detection of West Nile Virus

2020 ◽  
Vol 13 (10) ◽  
Author(s):  
Jae Woong Lee ◽  
Yu-Jung Won ◽  
Sung-Geun Lee ◽  
Soon-Young Paik
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Genomic Sequences ◽  
The Novel ◽  
Rt Pcr ◽  
West Nile ◽  
The Real ◽  
E Gene

Background: The West Nile Virus (WNV), discovered in New York, USA in 1999 after it was first isolated in Uganda in 1937, has since spread not only in the United States but also around the world. Africa, Eurasia, Australia, and the Middle East have sporadic cases of the disease. Objectives: We aimed to find real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to be more sensitive than conventional RT-PCR, and more rapid and efficient than conventional RT-PCR and real-time RT-PCR for WNV detection. Methods: A total of 32 genomic sequences from different strains of WNV were analyzed to identify conserved nucleotide sequence regions. Six WNV specific RT-LAMP primers targeting the E gene were designed. Results: The novel primer for the real-time RT-LAMP assay can detect WNV with high specificity. The efficiency of the real-time RT-LAMP assay is higher than the conventional RT-PCR and real-time RT-PCR. Real-time RT-PCR and conventional PCR require at least 30 – 40 min and 2 h, respectively, to yield results, whereas real-time RT-LAMP provides positive results in only 10 – 20 min. Conclusions: The novel primers were developed by analyzing of 32 genomic sequences of WNV strains. The primers were designed from the most conserved region of the E gene for real-time RT-LAMP. The LAMP assay is a rapid, efficient, highly sensitive, and specific tool for the identification of WNV.

scholarly journals Real-Time RT-PCR Assays for Detection and Genotyping of West Nile Virus Lineages Circulating in Africa

2016 ◽  
Vol 16 (12) ◽  
pp. 781-789 ◽  
Author(s):  
Gamou Fall ◽  
Martin Faye ◽  
Manfred Weidmann ◽  
Marco Kaiser ◽  
Anne Dupressoir ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Rt Pcr ◽  
West Nile ◽  
Pcr Assays

scholarly journals Laboratory and Field Evaluations of a Commercially Available Real-Time Loop-Mediated Isothermal Amplification Assay for the Detection of West Nile Virus in Mosquito Pools

2021 ◽  
Vol 37 (4) ◽  
pp. 256-262
Author(s):  
Kristen L. Burkhalter ◽  
Michael O'Keefe ◽  
Zachary Holbert-Watson ◽  
Theodore Green ◽  
Harry M. Savage ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Rt Pcr ◽  
Vector Surveillance ◽  
West Nile ◽  
Loop Mediated Isothermal Amplification ◽  
Surveillance Programs ◽  
Kappa Agreement

ABSTRACT Although the specific cDNA amplification mechanisms of reverse-transcriptase polymerase chain reaction (RT-PCR) and RT loop-mediated isothermal amplification (RT-LAMP) are very different, both molecular assays serve as options to detect arboviral RNA in mosquito pools. Like RT-PCR, RT-LAMP uses a reverse transcription step to synthesize complementary DNA (cDNA) from an RNA template and then uses target-specific primers to amplify cDNA to detectable levels in a single-tube reaction. Using laboratory-generated West Nile virus (WNV) samples and field-collected mosquito pools, we evaluated the sensitivity and specificity of a commercially available WNV real-time RT-LAMP assay (Pro-AmpRT™ WNV; Pro-Lab Diagnostics, Inc., Round Rock, Texas) and compared the results to a validated real-time RT-PCR assay. Laboratory generated virus stock samples containing ≥ 2.3 log10 plaque-forming units (PFU)/ml and intrathoracically inoculated mosquitoes containing ≥ 2.4 log10 PFU/ml produced positive results in the Pro-AmpRT WNV assay. Of field-collected pools that were WNV positive by real-time RT-PCR, 74.5% (70 of 94) were also positive by the Pro-AmpRT WNV assay, resulting in an overall Cohen's kappa agreement of 79.4% between the 2 tests. The Pro-AmpRT WNV assay shows promise as a suitable virus screening tool for vector surveillance programs provided agencies are aware of its characteristics and limitations.

scholarly journals Evaluation of West Nile Virus Diagnostic Capacities in Veterinary Laboratories of the Mediterranean and Black Sea Regions

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1038
Author(s):  
Elisa Pérez-Ramírez ◽  
Cristina Cano-Gómez ◽  
Francisco Llorente ◽  
Ani Vodica ◽  
Ljubiša Veljović ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Black Sea ◽  
Simultaneous Detection ◽  
Mediterranean Area ◽  
Rt Pcr ◽  
West Nile ◽  
Detection Rates ◽  
Animal Populations ◽  
Surveillance Programs ◽  
L1 And L2

The increasing incidence of West Nile virus (WNV) in the Euro-Mediterranean area warrants the implementation of effective surveillance programs in animals. A crucial step in the fight against the disease is the evaluation of the capacity of the veterinary labs to accurately detect the infection in animal populations. In this context, the animal virology network of the MediLabSecure project organized an external quality assessment (EQA) to evaluate the WNV molecular and serological diagnostic capacities of beneficiary veterinary labs. Laboratories from 17 Mediterranean and Black Sea countries participated. The results of the triplex real time RT-PCR for simultaneous detection and differentiation of WNV lineage 1 (L1), lineage 2 (L2) and Usutu virus (USUV) were highly satisfactory, especially for L1 and L2, with detection rates of 97.9% and 100%, respectively. For USUV, 75% of the labs reported correct results. More limitations were observed for the generic detection of flaviviruses using conventional reverse-transcription polymerase chain reaction (RT-PCR), since only 46.1% reported correct results in the whole panel. As regards the serological panel, the results were excellent for the generic detection of WNV antibodies. More variability was observed for the specific detection of IgM antibodies with a higher percentage of incorrect results mainly in samples with low titers. This EQA provides a good overview of the WNV (and USUV) diagnostic performance of the involved veterinary labs and demonstrates that the implemented training program was successful in upgrading their diagnostic capacities.

scholarly journals Co‐Infections: Simultaneous Detections of West Nile Virus and Usutu Virus in Birds from Germany

2021 ◽  
Author(s):  
Pauline Dianne Santos ◽  
Friederike Michel ◽  
Claudia Wylezich ◽  
Dirk Höper ◽  
Markus Keller ◽  
...  
Keyword(s):  
West Nile Virus ◽  
West Nile ◽  
Usutu Virus
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