Comparison of a novel real-time PCR method (RTA) and Artus RG for the quantification of HBV DNA and HCV RNA

Introduction: The purpose of this study was to evaluate and compare two manual isolation and real-time (RT) polymerase chain reaction (PCR) kits (RTA RT-PCR with RTA isolation kit and Artus RG RT-PCR with QIAamp isolation kit) for molecular diagnosis of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Methodology: The study was conducted on 121 and 54 clinical samples for the detection of HBV DNA and HCV RNA, respectively, with an additional 8 HCV RNA external quality control samples. Results: Though a high correlation was observed between the two kits for the HBV DNA (r = 0.955, p = 0.001) and HCV RNA quantifications (r = 0.828, p = 0.001), discordant results were found in nine of the HBV DNA and in six of the HCV RNA samples. The mean difference between the two systems was found to be 0.4 log IU/mL in Quality Control for Molecular Diagnostics (QCMD) HCV RNA samples by Bland-Altman analysis. Conclusions: Although there was a high correlation between HBV DNA and HCV RNA tests according to the results of the study, the RTA system requires improvement for the determination of HCV RNA.

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METHOD FOR FMD VIRUS 146S COMPONENT CONCENTRATION DETERMINATION WITH REAL-TIME REVERSE TRANSCRIPTION – POLYMERASE CHAIN REACTION IN VACCINE RAW MATERIALS

Veterinary Science Today ◽

10.29326/2304-196x-2018-2-25-31-35 ◽

2018 ◽

pp. 31-35

Author(s):

M. I. Doronin ◽

A. M. Timina ◽

D. A. Lozovoy ◽

V. A. Starikov ◽

D. V. Mikhalishin ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Regression Model ◽

Real Time ◽

Reverse Transcription ◽

Raw Materials ◽

Rt Pcr ◽

Chain Reaction ◽

Pcr Method ◽

Polymerase Chain

Method for determination of foot-and-mouth disease (FMD) virus 146S component concentration with real-time reverse transcription – polymerase chain reaction (rtRT-PCR) in vaccine raw materials is developed. Negative regression model of С146S = (30.269 – Ct)/4.155 type allowing determination of FMDV 146S particle concentrations based on the amplification threshold cycle values (Ct) is proposed. It has been experimentally proven that the quantity of the 146S component determined by the real-time RT-PCR method using developed regression model and contained in the inoculation dose of FMD vaccine confers protection to the vaccinated animals against generalized FMD of A, O, Asia-1 types. rtRT-PCR method is highly sensitive and allows rapid and highly reliable estimation of the 146S antigen concentration in FMD vaccine. The method for 146S particle quantity determination by real-time RT-PCR using the regression model is reliable and demonstrates high correlation (95.5–99.0%) with the complement fixation test results.

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METHOD FOR FMD VIRUS 146S COMPONENT CONCENTRATION DETERMINATION WITH REAL-TIME REVERSE TRANSCRIPTION – POLYMERASE CHAIN REACTION IN VACCINE RAW MATERIALS

Veterinary Science Today ◽

10.29326/2304-196x-2018-2-25-26-30 ◽

2018 ◽

pp. 26-30 ◽

Cited By ~ 1

Author(s):

M. I. Doronin ◽

A. M. Timina ◽

D. A. Lozovoy ◽

V. A. Starikov ◽

D. V. Mikhalishin ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Regression Model ◽

Real Time ◽

Reverse Transcription ◽

Raw Materials ◽

Rt Pcr ◽

Chain Reaction ◽

Pcr Method ◽

Polymerase Chain

Method for determination of foot-and-mouth disease (FMD) virus 146S component concentration with real-time reverse transcription – polymerase chain reaction (rtRT-PCR) in vaccine raw materials is developed. Negative regression model of С146S = (30.269 – Ct )/4.155 type allowing determination of FMDV 146S particle concentrations based on the amplification threshold cycle values (Ct ) is proposed. It has been experimentally proven that the quantity of the 146S component determined by the real-time RT-PCR method using developed regression model and contained in the inoculation dose of FMD vaccine confers protection to the vaccinated animals against generalized FMD of A, O, Asia-1 types. rtRT-PCR method is highly sensitive and allows rapid and highly reliable estimation of the 146S antigen concentration in FMD vaccine. The method for 146S particle quantity determination by real-time RT-PCR using the regression model is reliable and demonstrates high correlation (95.5–99.0%) with the complement fixation test results.

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Prevalence of occult hepatitis B virus infection among blood donors in Ouagadougou, Burkina Faso

African Journal of Clinical and Experimental Microbiology ◽

10.4314/ajcem.v22i3.7 ◽

2021 ◽

Vol 22 (3) ◽

pp. 359-364

Author(s):

A. Ky/Ba ◽

M. Sanou ◽

A.S. Ouédraogo ◽

I.B. Sourabié ◽

A.Y. Ky ◽

...

Keyword(s):

Hepatitis B ◽

Burkina Faso ◽

Real Time ◽

Real Time Pcr ◽

Blood Donors ◽

Hbv Dna ◽

Rt Pcr ◽

Occult Hepatitis ◽

B Virus ◽

Polymerase Chain

Background: In Burkina Faso, the polymerase chain reaction (PCR) assay is not routinely used in the biological qualification of blood donations and this constitutes a risk factor for the transmission of occult hepatitis B virus (HBV) infection during blood transfusion. The objective of this study is to determine the prevalence of occult B infection (OBI) among blood donors for the purposes of improved blood safety in Burkina Faso. Methodology: A descriptive cross-sectional study of 300 HBsAg negative blood donors was conducted in the city of Ouagadougou, Burkina Faso from April to October 2020. Anti-HBc antibody was determined using the BOSON® brand rapid tests. HBV DNA was detected in 75 selected donors by real-time PCR (rt PCR) using the 7500 Fast Real Time PCR assay technique. Results: Of the 300 HBsAg negative donors, 208 (69.3%) were males while 92 (30.7%) were females, with average age of 30.18 years. Anti-HBc antibody was detected in 39 cases (13%). Of the 75 donor samples tested by rt PCR, 3 (4%) were positive for HBV DNA (occult B infection); 2 of which were anti-HBc antibody positive (seropositive OBI) while 1 was anti-HBc antibody negative (seronegative OBI). Conclusion: Given the prevalence of OBI of 4% in this study and its consequences in blood recipients, it appears necessary that in addition to the classic serological markers of hepatitis B, to test for the presence of HBV DNA among blood donors in order to improve transfusion safety. Keywords: Prevalence, Occult B infection; Blood donors, Ouagadougou. French title: Prévalence de l'infection occulte par le virus de l'hépatite B chez les donneurs de sang à Ouagadougou, Burkina Faso Contexte: Au Burkina Faso, la polymérase chain réaction (PCR) n’est pas utilisée lors de la qualification biologique des dons et cela constitue un facteur de risque de transmission de l’Infection Occulte du virus B (VHB) lors des transfusions sanguines. L’objectif de cette étude était de déterminer la prévalence de l’infection occulte B chez les donneurs de sang en vue d’une meilleure sécurité transfusionnelle. Méthodologie: Une étude transversale prospective, réalisée d’avril à octobre 2020 dans la ville de Ouagadougou incluant 300 donneurs de sang AgHBs négatif. L’anticorps anti HBc a été déterminé par les tests rapides de marque BOSON®. L’ADN du VHB a été recherché chez 75 donneurs par PCR en temps réel (rt PCR) avec le 7500 Fast Real Time PCR. Résultats: Parmi les 300 donneurs AgHBs négatifs, 208 (69,3%) étaient des hommes et 92 (30,7%) des femmes. L’âge moyen était de 30,18 ans. La recherche de l’Ac anti-HBc était positive dans 39 cas (13 %). Parmi les 75 échantillons passés à la rt PCR, 3 (4%) étaient positifs pour l’ADN du VHB. Sur les 3 cas d’ADN VHB positifs, 2 (66,67%) étaient positifs Ac anti HBc et 1 (33,33%) Ac anti HBc négatif. Conclusion: Compte tenu de la prévalence de l’infection occulte B et ses conséquences chez les donneurs de sang et chez les receveurs, il devient nécessaire de rechercher, en plus des marqueurs sérologiques classiques de l’hépatite B, l’ADN VHB pour une meilleure sécurité transfusionnelle. Mots clés: Prévalence, Infection occulte de l’HBV, Donneurs de sang, Ouagadougou

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Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

Archives of Pathology & Laboratory Medicine ◽

10.5858/134.3.444 ◽

2010 ◽

Vol 134 (3) ◽

pp. 444-448 ◽

Cited By ~ 1

Author(s):

Zhengming Gu ◽

Jianmin Pan ◽

Matthew J. Bankowski ◽

Randall T. Hayden

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Quantitative Polymerase Chain Reaction ◽

Bk Virus ◽

Clinical Samples ◽

Quantitative Detection ◽

Chain Reaction ◽

Pcr Method ◽

Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

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A new real-time RT-PCR method allowing reliable absolute quantification of human cytokine mRNA in paediatric malaria clinical samples

Journal of Eukaryotic Microbiology ◽

10.1111/j.1550-7408.2005.05202003_3_2.x ◽

2005 ◽

Vol 52 (2) ◽

pp. 28S-34S

Author(s):

P. BOEUF ◽

I. VIGAN ◽

D. JUBLOT ◽

J.-C. BARALE ◽

B. GOKA ◽

...

Keyword(s):

Real Time ◽

Absolute Quantification ◽

Clinical Samples ◽

Rt Pcr ◽

Cytokine Mrna ◽

Paediatric Malaria ◽

Pcr Method ◽

Human Cytokine

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Deteksi Plasmodium falciparum dengan menggunakan metode real-time polymerase chain reaction di daerah Likupang dan Bitung

Jurnal e-Biomedik ◽

10.35790/ebm.4.1.2016.11057 ◽

2016 ◽

Vol 4 (1) ◽

Author(s):

David C. Tooy ◽

Janno B. Bernadus ◽

Angle Sorisi

Keyword(s):

Polymerase Chain Reaction ◽

Plasmodium Falciparum ◽

Real Time ◽

Real Time Pcr ◽

18S Rrna ◽

Rt Pcr ◽

Chain Reaction ◽

Negative Results ◽

Pcr Method ◽

Polymerase Chain

Abstract: Malaria is one of the most important parasitic disease which is caused by Plasmodium spp. There are approximately 1,2 billion people in the world with high risk of getting malaria. Plasmodium falciparum (P. falciparum) is the cause of tropical malaria or falciparum malaria, and is responsible for most of the mortality rate. Currently, real-time polymerase chain reaction (RT-PCR) is being studied as an alterative of conventional malarian examination. Mangold et al reported that RT-PCR have 94.1% sensitivity and 100% specificity compared to microscopic examination in detecting P. falciparum. The aim of this research is to detect the presence of P. falciparum using RT-PCR in Likupang and Bitung region. This research were using descriptive design to find out the capability of real-time PCR method to detect P. falciparum in Likupang dan Bitung region. The researcher have examined 71 samples which are fulfill the research sample’s criteria. Postive results of P. falciparum found in 18 samples (25,3%) and negative results in 53 samples (74,6%) of total 71 samples with using RT-PCR. No positive results were found in samples from Likupang. There are positive result of P. falciparum in samples from Bitung. It is concluded that RT-PCR method can detect the presence of P. falciparum from the samples obtained from Likupang and Bitung based on the presence of its DNA. This detection efford is done by using 18S rRNA as target gene and ajust specific temperature on the RT-PCR instrument.Keywords: Plasmodium falciparum, Real-time Polymerase Chain Reaction (PCR), DetectionAbstrak: Malaria merupakan salah satu penyakit penting yang disebabkan oleh parasit Plasmodium spp. Kira-kira 1,2 miliar penduduk dunia memiliki risiko tinggi untuk mendapat malaria. Di Indonesia sendiri, terdapat 343.527 kasus terkonfirmasi dan 45 kematian karena malaria. Plasmodium falciparum (P. Falciparum) merupakan penyebab dari malaria tropika atau malaria falsiparum, dan bertanggung jawab atas sebagian besar angka mortalitas. Saat ini Real-Time Polymerase Chain Reaction (RT-PCR) telah banyak diteliti sebagai alternatif dari pemeriksaan malaria. Mangold dkk melaporkan bahwa real-time PCR memiliki nilai sensitivitas 94,1% dan nilai spesifisitas 100% terhadap pemeriksaan mikroskopis dalam mendeteksi P. falciparum. Penelitian bertujuan untuk mendeteksi P. falciparum dengan menggunakan RT-PCR di daerah Likupang dan Bitung. Penelitian ini menggunakan rancangan penelitian deskriptif untuk mengetahui kemampuan metode real-time PCR dalam mendeteksi P. falciparum di daerah Likupang dan Bitung. Tujuan penelitian ini ialah untuk mendeteksi keberadaan P. falciparum dengan menggunakan metode real-time PCR di daerah Likupang dan Bitung. Peneliti memeriksa 71 sampel darah yang memenuhi kriteria sampel penelitian. Hasil positif P. falciparum ditemukan pada 18 sampel (25,3 %) dan hasil negatif pada 53 sampel (74,6 %) dari total 71 sampel dengan menggunakan RT-PCR. Tidak ditemukannya hasil positif P. falciparum pada sampel dari Likupang. Ditemukan hasil positif P. falciparum pada sampel dari Bitung. Simpulan: Metode RT-PCR dapat mendeteksi P. falciparum berdasarkan keberadaan DNA-nya pada sampel yang diperoleh dari daerah Likupang dan Bitung. Deteksi ini berhasil dilakukan dengan menggunakan 18S rRNA sebagai gen target dan pengaturan suhu tertentu pada instrument RT-PCR.Kata kunci: P. falciparum, Real-time Polymerase Chain Reaction (PCR), Detection

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Serological and Molecular Detection of Hepatitis B virus among patients referred to Kurdistan Center for Hepatology and Gastroenterology in Sulaimani City/Kurdistan Region of Iraq

UHD Journal of Science and Technology ◽

10.21928/uhdjst.v4n2y2020.pp117-122 ◽

2020 ◽

Vol 4 (2) ◽

pp. 117

Author(s):

Raz Sirwan Abdulla ◽

Salih Ahmed Hama

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Hbv Dna ◽

Molecular Techniques ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

B Virus ◽

Polymerase Chain ◽

Pcr Techniques ◽

Positive Results

Hepatitis B virus infection is caused by the hepatitis B virus, a major global health problem. This infection can lead to chronic conditions, followed by cirrhosis and hepatocellular carcinoma (HCC). The current study was aimed to detect HBV using serological and molecular techniques. During 2019, 300 blood samples were collected from Kurdistan Center for Hepatology and Gastroenterology in Sulaimani city. Enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) techniques were used for the detection of HBsAg and HBV DNA, respectively. Obtained results were revealed that 92 out of 300 tested patients (30.66%) seropositive for HBsAg. Among 92 seropositive patients, 53 were shown positive results for HBV DNA by RT-PCR. Dental clinic visiting and dialysis were among the important risk factors for HBV transmission. The vast majority of positive results were among males. Smokers showed relatively high rates of positive results. One-third of the referred patients who had liver complaints were positive for HBsAg. More than half of the seropositive patients showed RT-PCR positive results. It was concluded that the molecular method (RT-PCR) is more sensitive and gives a more accurate result than serology (ELISA). Therefore, it can be used as a diagnostic tool for HBV detection.

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Systematic Review and Meta-analysis of Real-time Polymerase Chain Reaction assay for the detection of COVID-19 from clinical samples in low-and middle-income countries: Protocol

10.21203/rs.3.rs-960246/v1 ◽

2021 ◽

Author(s):

Emmanuel Oladipo Babafemi

Keyword(s):

Systematic Review ◽

Polymerase Chain Reaction ◽

Real Time ◽

Meta Analysis ◽

Clinical Samples ◽

Rt Pcr ◽

Chain Reaction ◽

Pcr Assay ◽

Middle Income ◽

Polymerase Chain

Abstract Background: COVID-19 has spread globally since its discovery in Hubei province, China in December 2019 and became pandemic in 2020. COVID-19 is a new betacoronavirus and a variant of severe acute respiratory syndrome coronavirus 2 (SARA- CoV-2). Rapid, accurate and reliable diagnosis of COVID-19 will prevent the spread and allow for appropriate management. The main objective of this systematic review is to identify, appraise and summarise the published evidence on the diagnostic performance and effectiveness of SARS-CoV-2 virus in the diagnosis of current or previous COVID-19 using real-time polymerase chain reaction (RT-PCR) assay in low-and middle-income countries (LMICs). Methods: We will search MEDLINE/PubMed, EMBASE, BIOSIS, LILACS, Cochrane Infectious Diseases Group Specialised Register (CIDG SR), Global Health, and CINAHL for published studies for the diagnosis of COVID-19 using real-time polymerase chain reaction assay in LMICs There will be no restriction regarding the language, date of publication, and publication status. We will include retrospective, cross-sectional and cohort observational studies will be included in the review. Selection of studies, data extraction and management, assessment of risk of bias, and quality of evidence will be performed by two independent reviewers (EB and BC). A third researcher (GM) will be consulted in case of discrepancies. Depending on the availability and quality of the data, a meta-analysis will be performed. Otherwise, findings will be qualitatively reported. Discussion: To our knowledge, this is the first systematic review and meta-analysis to assess the uptake of RT-PCR assay for SARS-CoV-2 detection from clinical samples in human in LMICs. This review will make available evidence on the uptake, accuracy, approach, and interpretation of results of this assay in the context of COVID-19 diagnosis which will meet an urgent need, considering the diagnostic challenges of RT-PCR assay for COVID-19 diagnosis in humans. Systematic review registration: PROSPERO CRD42021271894

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Real Time PCR HBV-DNA Analysis in HBsAg Positive Patients

Journal of Armed Forces Medical College Bangladesh ◽

10.3329/jafmc.v10i2.25931 ◽

2015 ◽

Vol 10 (2) ◽

pp. 95-99

Author(s):

Wasila Rahman ◽

Muhammad Rabiul Hossain ◽

Arif Ahmed Khan ◽

Debashish Saha ◽

SM Mahbubul Alam ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Antiviral Therapy ◽

Real Time ◽

Real Time Pcr ◽

Armed Forces ◽

Hbv Dna ◽

Health Concern ◽

B Virus ◽

Pcr Method

Introduction: The hepatitis B virus is a global public health concern and leading cause of chronic liver disease in Bangladesh. For the diagnosis and monitoring of treatment of Hepatitis B virus infection, HBV-DNA detection and quantification is now extensively used worldwide.Objectives: The objective of this study was to detect HBV-DNA by real time PCR method in HBsAg positive patients, to compare the results of HBVDNA detection with HBeAg and Anti-HBe and to monitor the response after antiviral therapy in chronic hepatitis B patients and also to observe the intensity of hepatitis B infection in relation to age and sex.Methods: This was a cross sectional type of study conducted in Armed Forces Institute of Pathology (AFIP), Dhaka Cantonment. In this study, 56 sera of HBsAg positive patients were selected who all were subjected to do HBV-DNA (real time PCR) analysis during the period of 29 July to 30 0ctober, 2013.Results: Out of 56 HBsAg positive patients, HBV-DNA was detected in 34 patients. Among these, 8 (23.5%) patients were HBeAg positive, 16 (47%) patients were anti-HBe positive and 10 (29.5%) were negative for both HBeAg and anti-HBe. Age limit of patients was up to 60 years. HBV-DNA positive patients showed male predominance; 26 (76.5%) patients were male and 8 (23.5%) patients were female. Mean age of the patients was 35±14 years. Among 56 HBsAg positive patients, fifteen were receiving antiviral therapy. Out of them, HBV-DNA was decreased among 4 patients and could not be detected among 11 patients.Conclusion: Real time PCR method of detection of HBV-DNA is very important in patients who are HBeAg negative and this method is also applied to monitor treatment response to antivirals and to detect occult HBV infections immune control phase and also to detect reactivation of HBV cases.Journal of Armed Forces Medical College Bangladesh Vol.10(2) 2014

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