A panel of real-time PCR assays for the detection of Bourbon virus, Heartland virus, West Nile virus, and Trypanosoma cruzi in major disease–transmitting vectors

2021 ◽  
pp. 104063872110395
Author(s):  
Anushri Warang ◽  
Michael Zhang ◽  
Shuping Zhang ◽  
Zhenyu Shen
Keyword(s):  
West Nile Virus ◽  
Trypanosoma Cruzi ◽  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Animal Health ◽  
West Nile ◽  
Pcr Assays ◽  
Analytical Sensitivities ◽  
Pathogen Surveillance

Vector-borne pathogens, such as Bourbon virus (BRBV), Heartland virus (HRTV), West Nile virus (WNV), and Trypanosoma cruzi (TCZ) are a great threat to public health and animal health. We developed a panel of TaqMan real-time PCR assays for pathogen surveillance. PCR targets were selected based on nucleic acid sequences deposited in GenBank. Primers and probes were either designed de novo or selected from publications. The coverages and specificities of the primers and probes were extensively evaluated by performing BLAST searches. Synthetic DNA or RNA fragments (gBlocks) were used as PCR templates in initial assay development and PCR positive controls in subsequent assay validation. For operational efficiency, the same thermocycling profile was used in BRBV, HRTV, and WNV reverse-transcription quantitative PCR (RT-qPCR) assays, and a similar thermocycling profile without the initial reverse-transcription step was used in TCZ qPCR. The assays were optimized by titrating primer and probe concentrations. The analytical sensitivities were 100, 100, 10, and 10 copies of gBlock per reaction for BRBV (Cq = 36.0 ± 0.7), HRTV (Cq = 36.6 ± 0.9), WNV (Cq = 35.5 ± 0.4), and TCZ (Cq = 38.8 ± 0.3), respectively. PCR sensitivities for vector genomic DNA or RNA spiked with gBlock reached 100, 100, 10, and 10 copies per reaction for BRBV, HRTV, WNV, and TCZ, respectively. PCR specificity evaluated against a panel of non-target pathogens showed no significant cross-reactivity. Our BRBV, HRTV, WNV, and TCZ PCR panel could support epidemiologic studies and pathogen surveillance.

Detection of West Nile virus lineages 1 and 2 by real-time PCR

2007 ◽  
Vol 146 (1-2) ◽  
pp. 355-358 ◽  
Author(s):  
Sonja Linke ◽  
Heinz Ellerbrok ◽  
Matthias Niedrig ◽  
Andreas Nitsche ◽  
Georg Pauli
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
West Nile

Target Site Insensitivity Detection in Deltamethrin Resistant Culex pipiens Complex in Iran

2020 ◽  
Author(s):  
Reza ZEIDABADINEZHAD ◽  
Hassan VATANDOOST ◽  
Mohammad Reza ABAI ◽  
Navid DINPARAST DJADID ◽  
Abbasali RAZ ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Insecticide Resistance ◽  
Real Time ◽  
Real Time Pcr ◽  
Culex Pipiens ◽  
Mosquito Species ◽  
Knockdown Resistance ◽  
West Nile ◽  
Culex Pipiens Complex ◽  
Novel Approach

Background: Some mosquito species which belong to the Culex. pipiens complex are primary vectors for West Nile virus, Sindbis, Dirofilaria immitis, and many arboviruses. Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) gene of Cx. pipiens that is inherited, is one of the important threats for the efficacy of pyrethroids insecticides. Knockdown resistance (kdr) mutation, L1014F, is a well-defined mechanism of resistance to pyrethroids and DDT in many insect species. The aim of study was to determine the mechanisms of Insecticide resistance in this species Methods: Specimens of Cx. pipiens, the major vector of West Nile virus, were obtained in Tehran, Iran by collecting larvae from polluted wastewater in Qarchak of Tehran. In 2016 Insecticide susceptibility tests were performed according to WHO methods with deltamethrin 0.05%. We focused on determination of this point mutation in the VGSC gene of Cx. pipiens by Real-time PCR. Results: Our results revealed high levels of resistance to deltamethrin 0.05%. The lethal times i.e. LT50 and LT90 for deltamethrin were 2.1530 and 8.5117 h respectively. The result of Real-time PCR confirmed the presence of resistant genotype in all the members of tested population. This study is the first report on kdr genotyping of Cx. pipiens from Tehran and our results on the VGSC gene in position L1014F confirmed the TTA to TTT nucleotide change. Conclusion: This finding will provide a clue for management of insecticide resistance in mosquito which are vectors of arboviruses and decision for replacement of novel approach for vector control.

scholarly journals NS1 Protein Secretion during the Acute Phase of West Nile Virus Infection

2005 ◽  
Vol 79 (22) ◽  
pp. 13924-13933 ◽  
Author(s):  
Joanne Macdonald ◽  
Jessica Tonry ◽  
Roy A. Hall ◽  
Brent Williams ◽  
Gustavo Palacios ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Marker ◽  
Nonstructural Protein ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ns1 Protein ◽  
West Nile ◽  
Time Period

ABSTRACT The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

scholarly journals Development and Evaluation of Real-Time Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid and Sensitive Detection of West Nile Virus in Human Clinical Samples

2021 ◽  
Vol 10 ◽  
Author(s):  
Priyanka Singh Tomar ◽  
Sanjay Kumar ◽  
Sapan Patel ◽  
Jyoti S. Kumar
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Reverse Transcription ◽  
Febrile Illness ◽  
Posterior Uveitis ◽  
Qpcr Assay ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
West Nile Fever ◽  
West Nile

West Nile virus (WNV) causes West Nile fever and encephalitis worldwide. Currently, there are no effective drugs or vaccines available in the market to treat WNV infection in humans. Hence, it is of paramount importance to detect WNV early for the success of the disease control programs and timely clinical management in endemic areas. In the present paper, we report the development of real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for rapid and real-time detection of WNV targeting the envelope (env) gene of the virus. The RPA reaction was performed successfully at 39°C for 15 min in a real-time thermal cycler. The sensitivity of this assay was found similar to that of the quantitative real-time RT PCR (RT-qPCR) assay, which could detect 10 copies of the gene. The efficacy of the assay was evaluated with a panel of 110 WN suspected human samples showing the signs of retinitis, febrile illness and acute posterior uveitis. In comparison with RT-qPCR, RT-RPA showed a specificity of 100% (CI, 95.07–100%) and sensitivity of 96.15% (CI, 80.36–99.90%) with a negative (NPV) and positive predictive value (PPV) of 98.65 and 100%, respectively. The level of agreement between RT-RPA and reference RT-qPCR assay was shown to be very high. The turnaround time of real-time RPA assay is about 10-20 times faster than the RT-qPCR, which confirms its utility in the rapid and sensitive diagnosis of WNV infection. To the best of our knowledge, this is the first report which deals with the development of real-time RT-RPA assay for simple, rapid, sensitive, and specific detection of WNV in human clinical samples. The present RT-RPA assay proves to be a powerful tool that can be used for the rapid diagnosis of a large number of patient samples in endemic settings.

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
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