scholarly journals Optimization Of Real-Time Reverse Transcriptase Polymerase Chain Reaction For Detection Of Dengue Virus

2020 ◽  
Author(s):  
Kaunara Ally Azizi ◽  
Arnold J Ndaro ◽  
Athanasia Maro ◽  
Adonira T Saro ◽  
Reginald Kavishe
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Dengue Virus ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Diagnostic Methods ◽  
Virus Infections ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Objective Rapid and accurate laboratory confirmatory is very essential for control measures of dengue virus infections. However, many cases of dengue virus infections in most of the hospitals remain undiagnosed due to presence of other febrile illnesses with overlapping symptoms and lack of specificity in most of laboratory diagnostic methods. This study was set to optimize conditions for real time reverse transcriptase polymerase chain reaction (RT-PCR) for detection of dengue virus by using rapid and simple nucleic acid extraction method.Results The real time RT-PCR technique was successfully optimized using simple and rapid method for purification of nucleic acid, ‘boom method’. The technique works better when performed in a two-step procedure and can works well with all range of real time PCR machines. The optimized real time RT-PCR used in the present study is a valuable and reliable technique for routine diagnosis of dengue. Further investigation on the cost effectiveness in adopting this technique for routine screening and monitoring of the dengue infection should be done.

scholarly journals Optimization of Real-time Reverse Transcriptase Polymerase Chain Reaction for Detection of Dengue Virus

2020 ◽  
pp. 1-7
Author(s):  
Kaunara A. Azizi ◽  
Arnold J. Ndaro ◽  
Athanasia Maro ◽  
Adonira Saro ◽  
Reginald A. Kavishe
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Dengue Virus ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Reliable Technique ◽  
Polymerase Chain

Aims: This study was set to optimize conditions for real time reverse transcriptase polymerase chain reaction (RT-PCR) for detection of dengue virus by using rapid and simple nucleic acid extraction method. Methodology: One step and two step real time RT-PCR were evaluated in different PCR thermocyclers. Extraction of viral RNA was done by using a simple boom method. Results: The real time RT-PCR technique was successfully optimized using simple and rapid method for purification of nucleic acid, ‘boom method’. The technique works better when performed in a two-step procedure and can works well with all range of real time PCR machines. The optimized real time RT-PCR used in the present study is a valuable and reliable technique for routine diagnosis of dengue. Further investigation on the cost effectiveness in adopting this technique for routine screening and monitoring of the dengue infection should be done.

scholarly journals Use of immunodot blot and multiplex reverse transcriptase-polymerase chain reaction in dengue virus detection in macerates of Aedes aegypti larvae

2012 ◽  
Vol 3 (1) ◽  
pp. 13
Author(s):  
Aline T.A. Chagas ◽  
Michelle D. Oliveira ◽  
Jose M.S. Mezencio ◽  
Eduardo A.M. Silva ◽  
Leandro L. Oliveira ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Aedes Aegypti ◽  
Dengue Virus ◽  
Reverse Transcriptase ◽  
Epidemiological Surveillance ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Denv Serotypes ◽  
Polymerase Chain ◽  
Pcr Techniques

The <em>Dengue virus</em> is the main arbovirus that affects man in terms of morbidity and mortality. The detection of the virus is very important for epidemiological surveillance, so here we propose to standardize and compare the immunodot blot (IDB) and multiplex reverse transcriptase-polymerase chain reaction (M-RT-PCR) techniques to detect and characterize the dengue virus (DENV) serotypes in samples of <em>Aedes aegypti</em> larvae. Thus, the IDB and M-RT-PCR techniques were standardized using macerated samples of larvae collected in nature. The use of monoclonal antibodies in IDB has not shown great results, but DENV detection through this method was possible using polyclonal antibodies. The distinction of serotypes 1, 2 and 3 was carried out by M-RT-PCR.

Accuracy of real-time polymerase chain reaction to detect Schistosoma mansoni – infected individuals from an endemic area with low parasite loads

Parasitology ◽  
2020 ◽  
Vol 147 (10) ◽  
pp. 1140-1148
Author(s):  
Fernanda do Carmo Magalhães ◽  
Samira Diniz Resende ◽  
Carolina Senra ◽  
Carlos Graeff-Teixeira ◽  
Martin Johannes Enk ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Schistosoma Mansoni ◽  
Real Time ◽  
Diagnostic Methods ◽  
Control Measures ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Cross Sectional ◽  
Intestinal Schistosomiasis ◽  
Polymerase Chain

AbstractDue to the efforts to control schistosomiasis transmission in tropical countries, a large proportion of individuals from endemic areas present low parasite loads, which hinders diagnosis of intestinal schistosomiasis by the Kato-Katz (KK) method. Therefore, the development of more sensitive diagnostic methods is essential for efficient control measures. The aim was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) to detect Schistosoma mansoni DNA in fecal samples of individuals with low parasite loads. A cross-sectional population-based study was conducted in a rural community (n = 257) in Brazil. POC-CCA® was performed in urine and feces were used for RT-PCR. In addition, fecal exams were completed by 18 KK slides, saline gradient and Helmintex techniques. The combined results of the three parasitological tests detected schistosome eggs in 118 participants (45.9%) and composed the consolidated reference standard (CRS). By RT-PCR, 117 out of 215 tested samples were positive, showing 91.4% sensitivity, 80.2% specificity and good concordance with the CRS (kappa = 0.71). RT-PCR identified 86.9% of the individuals eliminating less than 12 eggs/g of feces, demonstrating much better performance than POC-CCA® (50.8%). Our results showed that RT-PCR is a valuable alternative for the diagnosis of intestinal schistosomiasis in individuals with very low parasite loads.

scholarly journals Real-time fluorogenic reverse transcription polymerase chain reaction assay for the specific detection of Bagaza virus

2012 ◽  
Vol 24 (5) ◽  
pp. 959-963 ◽  
Author(s):  
Dolores Buitrago ◽  
Ana Rocha ◽  
Cristina Tena-Tomás ◽  
Marta Vigo ◽  
Montserrat Agüero ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Diagnostic Methods ◽  
Nucleotide Sequencing ◽  
Alectoris Rufa ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Field Samples ◽  
Polymerase Chain

In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5’-Taq nuclease-3’ minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses ( Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak ( n = 11), as well as samples collected from partridges during surveillance programs ( n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.

scholarly journals Nucleic Acid Testing of SARS-CoV-2

2021 ◽  
Vol 22 (11) ◽  
pp. 6150
Author(s):  
Hee-Min Yoo ◽  
Il-Hwan Kim ◽  
Seil Kim
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Reverse Transcription ◽  
Diagnostic Methods ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Nucleic Acid Testing ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.

scholarly journals IDENTIFICATION OF DENGUE VIRUS SEROTYPES AT THE DR. SOETOMO HOSPITAL SURABAYA IN 2016 AND ITS CORRELATION WITH NS1 ANTIGEN DETECTION

2018 ◽  
Vol 23 (2) ◽  
pp. 151
Author(s):  
Jeine Stela Akualing ◽  
Aryati Aryati ◽  
Puspa Wardhani ◽  
Usman Hadi
Keyword(s):  
Polymerase Chain Reaction ◽  
Dengue Virus ◽  
Reverse Transcriptase ◽  
Ribonucleic Acid ◽  
Rna Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Ns1 Antigen ◽  
Dengue Virus Serotypes ◽  
Polymerase Chain

Serotipe virus dengue yang beredar terus mengalami perubahan dan berbeda di setiap daerah. Pergeseran serotipe maupun genotipedi dalamnya, mempengaruhi terjadinya wabah dengue di berbagai negara. Perbedaan serotipe diduga bernasab dengan deteksi antigen(Ag) non-structural 1 (NS1), namun belum banyak penelitian yang mendukung hal tersebut. Penelitian potong lintang dikerjakan sejakFebruari-Agustus 2016 dan didapatkan 60 subjek infeksi virus dengue (IVD) dan 25 non-IVD. Ribonucleic acid (RNA) virus denguediperiksa di semua subjek menggunakan Simplexa Dengue Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)termasuk identifikasi serotipe virus dengue dan pemeriksaan NS1 menggunakan uji cepat NS1 Panbio. Perbedaan perbandingan variabelkategorikal dianalisis dengan uji Fisher Exact. Kenasaban antara serotipe dengan deteksi Ag NS1 dianalisis dengan Chi-Kuadrat. RNAvirus dengue terdeteksi di 43 dari 60 subjek IVD (71,7%). Serotipe terbanyak adalah DENV-3 (62,8%). Pergeseran dominasi serotipetelah terjadi di Surabaya, sebelumnya dari DENV-2 ke DENV-1 dan sekarang DENV-3, kemungkinan akibat mobilitas pejamu, transporvirus dan faktor geografis. Kepekaan uji cepat NS1 75% dan kekhasan 100%. Persentase deteksi NS1 antar serotipe berbeda bermakna(p=0,002). Deteksi NS1 lebih rendah pada DENV-1 dibandingkan DENV-2 (p=0,007) ataupun DENV-3 (p=0,003). Serotipe virusdengue bernasab dengan deteksi NS1 (p=0,005). Ciri serotipe maupun genotipe virus dengue kemungkinan mempengaruhi sekresiNS1. Telah terjadi pergeseran serotipe virus dengue di pasien IVD di Surabaya sehingga diperlukan surveillance berkesinambunganuntuk memperkirakan terjadinya wabah. Serotipe bernasab dengan deteksi NS1. Salah satu penyebab hasil negatif palsu NS1 adalahperbedaan serotipe.

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