scholarly journals Field-deployable molecular diagnostic platform for arbovirus detection in Aedes aegypti

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Natalie Rutkowski ◽  
Yuemei Dong ◽  
George Dimopoulos
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic System ◽  
Molecular Diagnostic ◽  
Infection Prevalence ◽  
The Novel ◽  
Genomic Rna ◽  
Viral Genomic Rna

Abstract Background Surveillance of mosquito infection status is critical for planning and deployment of proper mosquito control initiatives. Point-of-care (POC) detection assays are necessary for monitoring the infection prevalence and geographical range of viruses in mosquito vector populations. We therefore assessed the novel real-time PCR (qPCR) bCUBE (Hyris, London, UK) molecular diagnostic system as a tool for virus detection. Methods Aedes aegypti Rps17 was used to validate and determine correlation coefficient for the novel bCUBE qPCR system to a laboratory standard StepOnePlus real-time PCR system (Applied Biosystems, Waltham, MA, USA). Experimentally infected Ae. aegypti were quantified for Zika (ZIKV) and dengue virus serotype 2 (DENV2) viral genomic RNA. Infection prevalence was compared to plaque assay. Results We developed and validated a novel qPCR system for the detection of ZIKV and DENV2 using the real-time qPCR system bCUBE. With bCUBE-based qRT-PCR, viral genomic RNA could be detected in individually infected Ae. aegypti mosquitoes and in pools of 5, 10 or 15 mosquitoes. Conclusions The portable qPCR bCUBE diagnostic system is capable of detecting Zika and dengue virus in mosquitoes and therefore has potential as a practical field-deployable diagnostic test for vector-borne disease surveillance programmes.

scholarly journals Field-deployable molecular diagnostic platform for arbovirus and Wolbachia detection in Aedes aegypti

2020 ◽  
Author(s):  
Natalie N. Rutkowski ◽  
Yuemei Dong ◽  
George Dimopoulos
Keyword(s):  
Dengue Virus ◽  
Disease Surveillance ◽  
Control Method ◽  
Mosquito Control ◽  
Point Of Care ◽  
Diagnostic System ◽  
Molecular Diagnostic ◽  
Infection Prevalence ◽  
Mosquito Vector ◽  
Virus Serotype

AbstractBackgroundSurveillance of mosquito infection status is critical for planning and deployment of proper mosquito control initiatives. Concurrently, Wolbachia is being widely used as a control method for arboviral transmission. Point-of-care (POC) detection assays are necessary for monitoring the infection prevalence and geographic range of viruses as well as Wolbachia in mosquito vector populations. We therefore assessed the novel qPCR bCUBE molecular diagnostic system as a tool for virus and Wolbachia detection.ResultsWe developed a reliable, specific, and sensitive diagnostic assay for detecting Zika virus and dengue virus serotype 2 using the real-time qPCR platform bCUBE. With bCUBE-based qRT-PCR, both Wolbachia bacterium and virus RNA could be reliably detected in individually infected Ae. aegypti mosquitoes and in pools of 5, 10, or 15 mosquitoes.ConclusionsThe portable qPCR bCUBE diagnostic platform is capable of detecting Zika and dengue virus as well as Wolbachia in mosquitoes and therefore has potential as a practical field-deployable diagnostic test for vector-borne disease surveillance programs.

Detection of dengue virus serotypes by single-tube multiplex RT-PCR and multiplex real-time PCR assay

2021 ◽  
Author(s):  
Kundan Tandel ◽  
Mahadevan Kumar ◽  
G.S. Bhalla ◽  
S.P.S. Shergill ◽  
Vijaya Swarnim ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Tube ◽  
Dengue Virus Serotypes

scholarly journals A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e89395 ◽  
Author(s):  
Jiyoun Yeo ◽  
Erin L. Crawford ◽  
Thomas M. Blomquist ◽  
Lauren M. Stanoszek ◽  
Rachel E. Dannemiller ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Testing ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Testing ◽  
Ffpe Samples ◽  
Pcr Method

Parasitological versus molecular diagnosis of strongyloidiasis in serial stool samples: how many?

2017 ◽  
Vol 92 (1) ◽  
pp. 12-16 ◽  
Author(s):  
E. Dacal ◽  
J.M. Saugar ◽  
T. Soler ◽  
J.M. Azcárate ◽  
M.S. Jiménez ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Correct Diagnosis ◽  
Molecular Techniques ◽  
Maximum Sensitivity ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Techniques ◽  
Molecular Technique ◽  
Asymptomatic Patients ◽  
Stool Samples

AbstractStrongyloidiasis is usually an asymptomatic disease in immunocompetent patients, caused by Strongyloides stercoralis. However, in immunocompromised patients it can produce a severe clinical profile. Therefore, a correct diagnosis is necessary in these cases and in those chronic asymptomatic patients. The low sensitivity of classical parasitological techniques requires the analysis of multiple serial stool samples. Molecular diagnostic techniques represent an improvement in the detection of the parasite. The objective of this study was to evaluate the minimum number of samples necessary to achieve maximum sensitivity by real-time polymerase chain reaction (PCR). A total of 116 stool samples from 39 patients were analysed by direct microscopic observation, agar culture, Harada–Mori and real-time PCR, in one, two, three and four or more consecutive samples. After two serial samples, 6 out of 39 patients were positive by parasitological and molecular techniques, while 16 of them were real-time PCR positive, and all the patients detected by parasitology were also detected by the molecular technique, reaching 100.00% sensitivity versus 83.00% when analysing a single sample. These data also reflect apparently low specificity (51.52%) and positive predictive value (PPV) (27.27 %) values, due to the high number of cases detected by real-time PCR and not by parasitological techniques. These cases were confirmed as true positives when analysing three, four or more samples from the same patient. In conclusion, the application of molecular techniques decreases the number of serial stool samples necessary to give a diagnosis with the maximum sensitivity.

scholarly journals Unexpected cases in field diagnosis of African swine fever virus in Vietnam: The needs consideration when performing molecular diagnostic tests

2020 ◽  
Vol 10 (2) ◽  
Author(s):  
Anh Duc Truong ◽  
Duc Viet Ly ◽  
Thi Hao Vu ◽  
Van Tuan Hoang ◽  
Thi Chinh Nguyen ◽  
...  
Keyword(s):  
Binding Site ◽  
Real Time ◽  
Real Time Pcr ◽  
Virus Isolation ◽  
False Negative ◽  
African Swine Fever ◽  
Molecular Diagnostic ◽  
Conventional Pcr ◽  
Sequencing Method ◽  
Single Mismatch

Background: The first confirmed case of African swine fever (ASF) in Vietnam was reported officially in February 2019. To date, ASF virus (ASFV) have been detected in 63/63 provinces in Vietnam. Currently, real-time polymerase chain reaction (PCR) is considered to be a powerful tool for viral detection in field samples, including ASFV. However, some recent reports have suggested that mismatches in primer and probe binding regions may directly affect real-time PCR qualification, leading a false-negative result.Aim: This study aims to further examine a conflicting result obtained from two OIE recommended methods, conventional PCR and real-time PCR, for ASFV detection.Methods: Two ASF suspected pigs from different provinces in the north of Vietnam were selected for this study based on clinical signs and postmortem lesions. The different results obtained by OIE-recommended conventional PCR and real-time PCR were further analyzed by the Sanger sequencing method and virus isolation in combination with hemadsorption (HAD) test using porcine alveolar macrophages cells.Results: The results showed that when the primer sequence matched perfectly with the sequences of field isolates, a mutation in probe binding region was found, indicating that a single mismatch in the probe binding site may cause a false-negative result by real-time PCR in detecting ASFV in clinical samples in Vietnam. An agreement between conventional PCR, using PPA1/PPA2 primers and two golden standard methods, virus isolation in combination with HAD assay, and sequencing method was observed in this study.Conclusion: A single mismatch in the probe binding site caused a failse-negative result by realtime PCR method in field diagnosis of ASFV. The needs consideration when selecting the appropriate molecular diagnostic methods is based on the current databases of ASFV sequences,  particularly for epidemiological surveillance of ASF. Keywords: African swine fever, PCR, Pigs, Real-time PCR, Vietnam

scholarly journals Performance of Three Commercial Molecular Diagnostic Assays for the Simultaneous Detection of Mycoplasma genitalium and Macrolide Resistance

2021 ◽  
Author(s):  
Chloé Le Roy ◽  
Cécile Bébéar ◽  
Sabine Pereyre
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Molecular Diagnostic ◽  
Handling Characteristics ◽  
Significant Difference ◽  
Commercial Kit ◽  
Resistance Detection

The increasing frequency of macrolide resistance is an emerging issue in the treatment of Mycoplasma genitalium infection. Because evaluation of new commercial kit detecting M. genitalium and macrolide resistance is needed, we evaluated the performance and handling characteristics of the Allplex MG & AziR (Seegene), the Macrolide-R/MG ELITe MGB (ELITechGroup), and the ResistancePlus MG FleXible kits (SpeeDx-Cepheid) in comparison with an in-house real-time PCR and 23S rRNA gene sequencing used as reference. A total of 239 urogenital specimens (135 M. genitalium-positive and 104 M. genitalium-negative specimens) collected between April and December 2019 at the French National Reference Center for bacterial Sexually Transmitted Infections were assessed. The overall agreement for M. genitalium detection of the three commercial kits compared with the in-house real-time PCR was 94.6–97.6%, and there was no significant difference. A total of 97 specimens were found M. genitalium-positive with the three kits and were used to assess macrolide resistance detection. The clinical sensitivity for resistance detection was 74.5% (95% confidence interval 61.7–84.2%), 96.2% (87.2–99.0%), and 92.8% (82.7–97.1%) for the Allplex MG & AziR, Macrolide-R/MG ELITe MGB, and ResistancePlusMG FleXible kits, respectively. The sensitivity of the Macrolide-R/MG ELITe MGB kit was significantly higher than that of the Allplex MG & AziR kit. The clinical specificity for resistance detection of the three kits was 97.4–97.6%. The random-access possibility, input sample volume, and DNA extract availability for detecting resistance to other antibiotics may also influence the selection of a commercial kit by diagnostic laboratories.

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