scholarly journals DO INDONESIAN GOVERNMENT DEPLOY RELIABLE AMMUNITION FOR COVID-19 MASS TEST? A COMPARISON OF REAL-TIME PCR KITS

2021 ◽  
pp. 75-77
Author(s):  
NELLY MARISSA ◽  
SALMIATY ◽  
SARI HANUM ◽  
EVAN FEBRIANSYAH ◽  
NUR RAMADHAN ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Great Ability ◽  
Commercial Kits ◽  
Mass Test ◽  
Amplification Procedure

Objective: This study aimed to compare the level of reliability of three commercial real-time PCR kits in determining clinical samples. Methods: A total of 40 swabs samples which were previously tested positive, were re-test using the BioCov-19 RT-PCR kit, Sansure coronavirus disease 19 (COVID-19) nucleic acid diagnostic kit, and Kogen PowerCheck with Thermocycler (Roche). The amplification procedure is carried out based on the manual for each kit. Results: Sansure COVID-19 nucleic acid diagnostic was able to detect 40 samples with positive severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) results detected in both genes, while the PowerChek™ 2019-nCoV real-time PCR kit able to detect 35 samples showed that SARS-COV-II was detected in both genes, and the BioCoV-19 RT-PCR Kit brand kit able to detect 34 samples showed positive SARS-COV-2 results in both genes. Conclusion: The three commercial kits show great ability detection, so that they can be used to detect the presence of SARS-COV-II in clinical samples, and also in mass screening.

scholarly journals The First Quarter of SARS-CoV-2 Testing: the University of Washington Medicine Experience

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Alexander L. Greninger ◽  
Keith R. Jerome
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Real Time Pcr ◽  
Medical Center ◽  
Rt Pcr ◽  
University Of Washington ◽  
Clinical Laboratories ◽  
To Come ◽  
The Many ◽  
The University

ABSTRACT In early March 2020, the University of Washington Medical Center clinical virology laboratory became one of the first clinical laboratories to offer testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). When we first began test development in mid-January, neither of us believed there would be more than 2 million confirmed SARS-CoV-2 infections nationwide or that we would have performed more than 150,000 real-time PCR (RT-PCR) tests, with many more to come. This article will be a chronological summary of how we rapidly validated tests for SARS-CoV-2, increased our testing capacity, and addressed the many problems that came up along the way.

scholarly journals Harmonization of Bordetella pertussis Real-Time PCR Diagnostics in the United States in 2012

2014 ◽  
Vol 53 (1) ◽  
pp. 118-123 ◽  
Author(s):  
Margaret M. Williams ◽  
Thomas H. Taylor ◽  
David M. Warshauer ◽  
Monte D. Martin ◽  
Ann M. Valley ◽  
...  
Keyword(s):  
Public Health ◽  
Real Time ◽  
Bordetella Pertussis ◽  
Real Time Pcr ◽  
The United States ◽  
Extraction Methods ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Content Type

Real-time PCR (rt-PCR) is an important diagnostic tool for the identification ofBordetella pertussis,Bordetella holmesii, andBordetella parapertussis. Most U.S. public health laboratories (USPHLs) target IS481, present in 218 to 238 copies in theB. pertussisgenome and 32 to 65 copies inB. holmesii. The CDC developed a multitarget PCR assay to differentiateB. pertussis,B. holmesii, andB. parapertussisand provided protocols and training to 19 USPHLs. The 2012 performance exercise (PE) assessed the capability of USPHLs to detect these threeBordetellaspecies in clinical samples. Laboratories were recruited by the Wisconsin State Proficiency Testing program through the Association of Public Health Laboratories, in partnership with the CDC. Spring and fall PE panels contained 12 samples each of viableBordetellaand non-Bordetellaspecies in saline. Fifty and 53 USPHLs participated in the spring and fall PEs, respectively, using a variety of nucleic acid extraction methods, PCR platforms, and assays. Ninety-six percent and 94% of laboratories targeted IS481in spring and fall, respectively, in either singleplex or multiplex assays. In spring and fall, respectively, 72% and 79% of USPHLs differentiatedB. pertussisandB. holmesiiand 68% and 72% identifiedB. parapertussis. IS481cycle threshold (CT) values forB. pertussissamples had coefficients of variation (CV) ranging from 10% to 28%. Of the USPHLs that differentiatedB. pertussisandB. holmesii, sensitivity was 96% and specificity was 95% for the combined panels. The 2012 PE demonstrated increased harmonization of rt-PCRBordetelladiagnostic protocols in USPHLs compared to that of the previous survey.

scholarly journals Evaluation of real-time nucleic acid sequence-based amplification for detection of Chikungunya virus in clinical samples

2009 ◽  
Vol 58 (9) ◽  
pp. 1168-1172 ◽  
Author(s):  
J.-N. Telles ◽  
K. Le Roux ◽  
P. Grivard ◽  
G. Vernet ◽  
A. Michault
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Chikungunya Virus ◽  
False Negative ◽  
Nucleic Acid Sequence ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Plasma Samples ◽  
Rt Pcr ◽  
Nucleic Acid Extraction

The Chikungunya virus (CHIKV) is a member of the genus Alphavirus that is transmitted to humans by Aedes mosquitoes. In 2005 and 2006, the Indian Ocean island of La Réunion was hit with an unprecedented CHIKV fever outbreak that infected 300 000 people. In the present study, we describe the evaluation of real-time nucleic acid sequence-based amplification (RT-NASBA) for the detection of CHIKV in clinical samples. A co-extracted and co-amplified chimerical CHIKV RNA sequence was used as an internal control to eliminate false-negative results. The detection threshold of the assay was determined from quantified CHIKV-positive plasma, and estimated to be 200 copies per NASBA reaction. The specificity of the assay was determined using blast analyses and non-cross-reactivity using an O'nyong-nyong virus culture and 250 CHIKV RT-PCR-negative plasma samples. A 100 % specificity was found and no invalid result was obtained, showing the good quality of the nucleic acid extraction. The assay was then evaluated using 252 CHIKV-positive RT-PCR plasma samples. The samples were all tested positive, including those with low viral load. This evaluation showed that the RT-NASBA is a rapid (5 h from sample nucleic acid extraction to detection), sensitive, specific and reliable method for the routine diagnosis of CHIKV in clinical samples.

scholarly journals Antiprimer Quenching-Based Real-Time PCR and Its Application to the Analysis of Clinical Cancer Samples

2006 ◽  
Vol 52 (4) ◽  
pp. 624-633 ◽  
Author(s):  
Jin Li ◽  
Fengfei Wang ◽  
Harvey Mamon ◽  
Matthew H Kulke ◽  
Lyndsay Harris ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Genetic Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Low Cost ◽  
Pcr Primers ◽  
Medical Diagnostics ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Correct Identification

Abstract Background: Nucleic acid amplification plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics, and drug discovery. We present a novel quantitative PCR technology that combines the advantages of existing methods and allows versatile and flexible nucleic acid target quantification in clinical samples of widely different origin and quality. Methods: We modified one of the 2 PCR primers by use of an oligonucleotide “tail” fluorescently labeled at the 5′ end. An oligonucleotide complementary to this tail, carrying a 3′ quenching molecule (antiprimer), was included in the reaction along with 2 primers. After primer extension, the reaction temperature was lowered such that the antiprimer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double-stranded PCR product. The latter provides real-time fluorescent product quantification. This antiprimer-based quantitative real-time PCR method (aQRT-PCR) was used to amplify and quantify minute amounts of input DNA for genes important to cancer. Results: Simplex and multiplex aQRT-PCR demonstrated linear correlation (r2 >0.995) down to a DNA input equivalent to 20 cells. Multiplex aQRT-PCR reliably identified the HER-2 gene in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA from cancer patients. Adaptation to multiplex single-nucleotide polymorphism detection via allele-specific aQRT-PCR allowed correct identification of apolipoprotein B polymorphisms in 51 of 51 human specimens. Conclusion: The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens.

scholarly journals Expression of PDCD1 (PD-1) Gene among Non-small Cell Lung Cancer (NSCLC) Patients with Real-Time PCR Application

2019 ◽  
pp. 1-9
Author(s):  
Seyed Mohammad Amin Ahmadpanah ◽  
Mohammadreza Ghanbari ◽  
Seyed Alireza Janani ◽  
Fahimeh Nemati
Keyword(s):  
Lung Cancer ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Lung Cancer Patients ◽  
Nsclc Patients ◽  
Age Range ◽  
Disease Grade ◽  
Main Ligand

Background: PD-L1 is the main ligand is expressed on many tumors including lung cancer and is expressed in hematopoietic cells and various leukemia. The aim of this study was to evaluate the expression of PD-1 gene and the evaluation of cancerous grades of NSCLC and its subclasses from lung cancer patients in Tehran hospitals using Real-Time PCR. Materials and Methods: A total of 35 clinical samples were collected from patients with NSCLC-derived lung cancer from three hospitals in Tehran (Khatam Hospital, Athiyah Hospital, and Masih Hospital). Of the 35 samples collected in 2017, 20% of the patients were women and 80% of them were male. The range of patients’ age spectrum was 37 - 80 years. The disease grade of the patients in this study was varied and 22 different grades among them. To investigate the PDCD-1 gene expression level, after extraction of RNA and cDNA synthesis the Real-Time PCR was done and the expression of the gene was investigated. Results: The highest grade was IIIa which contained 6 patients (17.1%). 74% of adenocarcinoma cases were in T-categories of lung cancer and 25% of patients were in grade IIIa. Patients with the grade of T3 were observed in 4 samples, 2 had adenocarcinoma and 2 with SCC with age range of 55 -62 years. The results showed that the expression of PDCD-1 increased 2.46 Fold more in patients with lung cancer than NSCLC. Conclusion: The results of this study showed that there is a significant relationship between the PDCD1 or PD-1 expression of NSCLC-type lung cancer compared with healthy individuals, and using the RT-PCR for ease and rapidity it can be proved.

scholarly journals Genome Sequence of a Minacovirus Strain from a Farmed Mink in The Netherlands

2021 ◽  
Vol 10 (8) ◽  
Author(s):  
Kirsty T. T. Kwok ◽  
Myrna M. T. de Rooij ◽  
Felisita F. Sinartio ◽  
Lidwien A. M. Smit ◽  
Marion P. G. Koopmans ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
The Netherlands ◽  
Real Time ◽  
Genome Sequence ◽  
Deep Sequencing ◽  
Real Time Pcr ◽  
Fecal Sample ◽  
Viral Genome ◽  
Rt Pcr ◽  
Content Type

ABSTRACT We report the genome sequence of a Minacovirus strain identified from a fecal sample from a farmed mink (Neovison vison) in The Netherlands that was tested negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using real-time PCR (RT-PCR). The viral genome sequence was obtained using agnostic deep sequencing.

Multiplex real-time PCR assays for the prediction of cephalosporin, ciprofloxacin and azithromycin antimicrobial susceptibility of positive Neisseria gonorrhoeae nucleic acid amplification test samples

2020 ◽  
Vol 75 (12) ◽  
pp. 3485-3490 ◽  
Author(s):  
S W Peterson ◽  
I Martin ◽  
W Demczuk ◽  
N Barairo ◽  
P Naidu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Real Time Pcr ◽  
Nucleic Acid Amplification ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Snp Analysis ◽  
Rt Pcr ◽  
Pcr Assays

Abstract Background The incidence of antimicrobial-resistant Neisseria gonorrhoeae (GC) is rising in Canada; however, antimicrobial resistance (AMR) surveillance data are unavailable for infections diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs), representing over 80% of diagnoses. We developed a set of 10 improved molecular assays for surveillance of GC-AMR and prediction of susceptibilities in NAAT specimens. Methods Multiplex real-time PCR (RT–PCR) assays were developed to detect SNPs associated with cephalosporin (ponA, porB, mtrR −35delA, penA A311V, penA A501, N513Y, G545S), ciprofloxacin (gyrA S91, parC D86/S87/S88) and azithromycin [23S (A2059G, C2611T), mtrR meningitidis-like promoter] resistance. The assays were validated on 127 gonococcal isolates, 51 non-gonococcal isolates and 50 NAATs with matched culture isolates. SNPs determined from the assay were compared with SNPs determined from in silico analysis of WGS data. MICs were determined for culture isolates using the agar dilution method. Results SNP analysis of the 50 NAAT specimens had 96% agreement with the matched culture RT–PCR analysis. When compared with MICs, presence of penA A311V or penA A501 and two or more other SNPs correlated with decreased susceptibility and presence of three or more other SNPs correlated with intermediate susceptibility to cephalosporins; presence of any associated SNP correlated with ciprofloxacin or azithromycin resistance. NAAT-AMR predictions correlated with matched-culture cephalosporin, ciprofloxacin and azithromycin MICs at 94%, 100% and 98%, respectively. Conclusions We expanded molecular tests for N. gonorrhoeae AMR prediction by adding new loci and multiplexing reactions to improve surveillance where culture isolates are unavailable.

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 Novel rapid identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by real-time RT-PCR using BD Max Open System in Taiwan

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9318 ◽  
Author(s):  
Cherng-Lih Perng ◽  
Ming-Jr Jian ◽  
Chih-Kai Chang ◽  
Jung-Chung Lin ◽  
Kuo-Ming Yeh ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Turnaround Time ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Respiratory Syndrome Virus ◽  
Acid Extraction ◽  
Prevention Measures ◽  
Rt Pcr ◽  
African Region

Coronavirus disease 2019 has become a worldwide pandemic. By April 7, 2020, approximately 1,279,722 confirmed cases were reported worldwide including those in Asia, European Region, African Region and Region of the Americas. Rapid and accurate detection of Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) is critical for patient care and implementing public health measures to control the spread of infection. In this study, we developed and validated a rapid total nucleic acid extraction method based on real‐time RT-PCR for reliable, high‐throughput identification of SARS-CoV-2 using the BD MAX platform. For clinical validation, 300 throat swab and 100 sputum clinical samples were examined by both the BD MAX platform and in-house real-time RT-PCR methods, which showed 100% concordant results. This BD MAX protocol is fully automated and the turnaround time from sample to results is approximately 2.5 h for 24 samples compared to 4.8 h by in-house real-time RT-PCR. Our developed BD MAX RT-PCR assay can accurately identify SARS-CoV-2 infection and shorten the turnaround time to increase the effectiveness of control and prevention measures for this emerging infectious disease.

scholarly journals Rapid detection of avian leukosis virus subgroup J by cross-priming amplification

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yong Xiang ◽  
Lizhen Li ◽  
Peng Liu ◽  
Ling Yan ◽  
Zeng Jiang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Cell Cultures ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Avian Leukosis Virus ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Subgroup J ◽  
Avian Leukosis Virus Subgroup

AbstractAvian leukosis virus subgroup J (ALV-J) causes oncogenic disease in chickens in China, resulting in great harm to poultry production, and remains widespread in China. Herein, we employed a cross-priming amplification (CPA) approach and a nucleic acid detection device to establish a visual rapid detection method for ALV-J. The sensitivity of CPA, polymerase chain reaction (PCR) and real-time PCR (RT-PCR) was compared, and the three methods were used to detect ALV-J in the cell cultures which inoculated with clinical plasma. The result showed when the amplification reaction was carried out at 60 °C for just 60 min, the sensitivity of CPA was 10 times higher than conventional PCR, with high specificity, which was comparable with RT-PCR, based on detection of 123 cell cultures which inoculated with clinical plasma, the coincidence rate with real-time PCR was 97.3% (71/73). CPA detection of ALV-J does not require an expensive PCR instrument; a simple water bath or incubator is sufficient for complete DNA amplification, and the closed nucleic acid detection device avoids aerosol pollution, making judgment of results more intuitive and objective. The CPA assay would be a promising simple, rapid and sensitive method for identification of ALV-J.

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