scholarly journals One-step real-time RT-PCR assays for serotyping dengue virus in clinical samples

2015 ◽  
Vol 15 (1) ◽  
Author(s):  
Erik Alm ◽  
Gunnel Lindegren ◽  
Kerstin Ingrid Falk ◽  
Nina Lagerqvist
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Clinical Samples ◽  
Rt Pcr ◽  
One Step ◽  
Pcr Assays

scholarly journals Performance Evaluation of Commercial Dengue Diagnostic Tests for Early Detection of Dengue in Clinical Samples

2017 ◽  
Vol 2017 ◽  
pp. 1-4 ◽  
Author(s):  
Tuan Nur Akmalina Mat Jusoh ◽  
Rafidah Hanim Shueb
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Diagnostic Test ◽  
Rapid Diagnostic Test ◽  
Dengue Infection ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Serum Samples ◽  
Kappa Agreement

The shattering rise in dengue virus infections globally has created a need for an accurate and validated rapid diagnostic test for this virus. Rapid diagnostic test (RDT) and reverse transcription-polymerase chain reaction (RT-PCR) diagnostic detection are useful tools for diagnosis of early dengue infection. We prospectively evaluated the diagnostic performance of nonstructural 1 (NS1) RDT and real-time RT-PCR diagnostic kits in 86 patient serum samples. Thirty-six samples were positive for dengue NS1 antigen while the remaining 50 were negative when tested with enzyme-linked immunosorbent assay (ELISA). Commercially available RDTs for NS1 detection, RTK ProDetect™, and SD Bioline showed high sensitivity of 94% and 89%, respectively, compared with ELISA. GenoAmp® Trioplex Real-Time RT-PCR and RealStar® Dengue RT-PCR tests presented a comparable kappa agreement with 0.722. The result obtained from GenoAmp® Real-Time RT-PCR Dengue test showed that 14 samples harbored dengue virus type 1 (DENV-1), 8 samples harbored DENV-2, 2 samples harbored DENV-3, and 1 sample harbored DENV-4. 1 sample had a double infection with DENV-1 and DENV-2. The NS1 RDTs and real-time RT-PCR tests were found to be a useful diagnostic for early and rapid diagnosis of acute dengue and an excellent surveillance tool in our battle against dengue.

scholarly journals Genetic Diversity of Porcine Reproductive and Respiratory Syndrome Virus and Evaluation of Three One-Step Real-Time RT-PCR Assays in Korea

2021 ◽  
Author(s):  
Go-Eun Shin ◽  
Ji-Young Park ◽  
Kyoung-Ki Lee ◽  
Mi-Kyeong Ko ◽  
Bok-Kyung Ku ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Real Time ◽  
Economic Losses ◽  
Respiratory Syndrome Virus ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
The Real ◽  
One Step ◽  
Pcr Assays

Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses in the global swine industry. Frequent genetic variations in this virus cause difficulties in controlling and accurately diagnosing PRRSV. Methods In this study, we investigated the genetic characteristics of PRRSV-1 and PRRSV-2 circulating in Korea from January 2018 to September 2021 and evaluated three one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Results A total of 129 lung samples were collected, consisting of 47 samples for PRRSV-1, 62 samples for PRRSV-2, and 20 PRRSV-negative samples. Nucleotide sequence analysis of open reading frames (ORFs) 5, ORF6, and ORF7 genes from PRRSV samples showed that PRRSV-1 belonged to subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%), whereas PRRSV-2 was classified as lineage 1 (25/62, 40.32%), Korean lineage (Kor) C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%). Amino acid sequence analysis showed that the neutralizing epitope and T cell epitope of PRRSV-1, and the decoy epitope region and hypervariable regions of PRRSV-2 had evolved under positive selection pressure. In particular, the key amino acid substitutions were found at positions 102 and 104 of glycoprotein 5 (GP5) in some PRRSV-2, and at positions 10 and 70 of membrane protein (M) in most PRRSV-2. In addition, one-step real-time RT-PCR assays, comprising two commercial tests and one test recommended by the World Organization for Animal Health (OIE), were evaluated. Conclusion The results revealed that two of the real-time RT-PCR assays had high sensitivities and specificities, whereas the real-time RT-PCR assay of the OIE had low sensitivity due to mismatches between nucleotides of Korean PRRSVs and forward primers. In this study, we genetically characterized recent PRRSV occurrences and evaluated three one-step real-time RT-PCR assays used in Korea.

scholarly journals Rapid Detection and Molecular Typing of Dengue Virus by Using Multiplex-Nested-RT-PCR

2015 ◽  
Vol 11 (2) ◽  
Author(s):  
Nastiti Wijayanti ◽  
Hera Nirwati ◽  
Tri Wibawa ◽  
Aris Haryanto ◽  
S. Sutaryo
Keyword(s):  
Dengue Virus ◽  
Nested Pcr ◽  
Genotypic Variation ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Virus Serotype ◽  
Pcr Multiplex ◽  
Pcr Products ◽  
One Step ◽  
Nested Pcr Assays

world. We have evaluated the combination of one-step RT-PCR and multiplex nested PCR assays for detectingdengue viruses from clinical samples. Twelve patients were screened for the dengue virus, using a pair of primersthat conserve for several Flavivirus. The results showed that in 12 suspect patients, 100% were positive for Flavivirusand there are some genotypic variation among them, that indicated by several RT-PCR products higher than 511 bp,the expected product for RT-PCR. Further assay was performed to clarify the presence and serotypes of dengue virususing multiplex nested PCR. Serotyping results indicated that 83,3% of samples can be confirmed for dengue virus.Among the dengue virus positive 16,7 % are dengue-2, 16.7 % are dengue-3, and the most common 50% are dengue-4,whereas dengue-1 were not found among the patients. The combination of RT-PCR and multiplex nested PCR assaycan be used for rapid analysis dengue samples in early phase which is potentially useful for clinical, epidemiologyand also evolutionary studies.Key words: Flavivirus, dengue virus, serotype, RT-PCR, multiplex nested PCR

scholarly journals One-step multiplex real-time RT-PCR for detection and typing of dengue virus

2019 ◽  
Vol 43 ◽  
pp. 86-91 ◽  
Author(s):  
Myung-Jin Mun ◽  
Joon-Yong Bae ◽  
Jin Hyuck Kim ◽  
Soo Bok Kim ◽  
Ilseob Lee ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Rt Pcr ◽  
One Step

Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease

2016 ◽  
Vol 235 ◽  
pp. 168-175 ◽  
Author(s):  
K. Gorna ◽  
A. Relmy ◽  
A. Romey ◽  
S. Zientara ◽  
S. Blaise-Boisseau ◽  
...  
Keyword(s):  
Real Time ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Rt Pcr ◽  
Foot And Mouth ◽  
One Step ◽  
Pcr Assays

A simple one-step real-time RT-PCR for diagnosis of dengue virus infection

2008 ◽  
Vol 80 (8) ◽  
pp. 1426-1433 ◽  
Author(s):  
Harryson Wings Godoy dos Santos ◽  
Telma Regina Ramos Silva Poloni ◽  
Kelly Paula Souza ◽  
Vanessa Danielle Menjon Muller ◽  
Flávia Tremeschin ◽  
...  
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
Real Time ◽  
Rt Pcr ◽  
Dengue Virus Infection ◽  
One Step

scholarly journals Real-time PCR for detecting circulating dengue virus in the Guangdong Province of China in 2006

2008 ◽  
Vol 57 (12) ◽  
pp. 1547-1552 ◽  
Author(s):  
Zhijun Bai ◽  
Licheng Liu ◽  
Zeng Tu ◽  
Lisi Yao ◽  
Jianwei Liu ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Sequencing Analysis ◽  
Serum Samples ◽  
Serological Tests ◽  
Wide Range ◽  
Pcr Assays

Dengue virus (DENV) causes a wide range of diseases in humans, from the acute febrile illness dengue fever (DF) to life-threatening dengue haemorrhagic fever/dengue shock syndrome. We developed four real-time quantitative PCR assays for each serotype of DENV based on computational analysis. These assays had high sensitivity and specificity without cross-reactivity for the four serotypes. To evaluate the performance of these assays in detecting and typing the virus in clinical samples, we analysed 64 serum samples from Guangdong during 2006. The results showed that 71 % of those samples were positive by the DEN-1 assay. The DENV assay results, in agreement with the serological tests and sequencing analysis, showed that the pathogen resulting in the DF explosion in Guangdong in 2006 belonged to DEN-1. Compared to the serological assays, the real-time PCR assays that we developed were much more sensitive in the 1–3 days after onset of the symptoms.

scholarly journals Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays

2004 ◽  
Vol 50 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Leo L M Poon ◽  
Kwok Hung Chan ◽  
On Kei Wong ◽  
Timothy K W Cheung ◽  
Iris Ng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Stool Samples ◽  
Pcr Assays

Abstract Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.

scholarly journals Individual-based dengue virus surveillance in Aedes aegypti mosquitoes collected concurrently with suspected patients in Tarlac City, Philippines

2020 ◽  
Author(s):  
Jean Claude Balingit ◽  
Thaddeus M. Carvajal ◽  
Mariko Saito-Obata ◽  
Maribet Gamboa ◽  
Amalea Dulcene Nicolasora ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Vector Control ◽  
Real Time ◽  
Phylogenetic Analyses ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Virus Surveillance ◽  
One Step ◽  
Mosquito Infection

Abstract Background: Vector control measures are critical in the prevention and reduction of dengue virus (DENV) transmission. In this context, effective vector control is reliant not only on knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito infection. Mosquito-based virus surveillance programs commonly rely on pool-based mosquito testing, but whether individual-based mosquito testing could represent a feasible alternative is not largely studied. Applying an individual-based mosquito testing approach, we conducted a one-month DENV surveillance of adult Aedes aegypti mosquitoes around households of suspected dengue patients during the 2015 dengue peak season in Tarlac City, Philippines to more accurately assess the mosquito infection rate, and to identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients. Methods: We performed a one-step multiplex real-time RT-PCR assay for the simultaneous detection and serotyping of DENV in patients and in individual female Ae. aegypti mosquito. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENVs in mosquitoes and patients at the genotype level. Results: We collected a total of 583 adult Ae. aegypti mosquitoes, of which we tested 359 female mosquitoes individually for the presence of the DENV. Ten mosquitoes (2.8%) from amongst 359 female mosquitoes were confirmed to be positive for the presence of the DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which were consistent with the serotypes concurrently infecting patients. Sequencing and phylogenetic analyses of the detected DENVs based on the partial envelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely: DENV-1 genotype IV, DENV-2 Cosmopolitan genotype and DENV-4 genotype II. Conclusions: In this study, we demonstrate the utility of a one-step multiplex real-time RT-PCR assay in individual-based DENV surveillance of mosquitoes. Our findings reinforce the importance of detecting and monitoring virus activity in local mosquito populations, which is critical for dengue prevention and control activities.

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