scholarly journals Harnessing recombinase polymerase amplification for rapid detection of SARS-CoV-2 in resource-limited settings

2021 ◽  
Author(s):  
Dounia Cherkaoui ◽  
Da Huang ◽  
Benjamin S. Miller ◽  
Rachel A. McKendry
Keyword(s):  
Real Time ◽  
Gold Standard ◽  
Simultaneous Detection ◽  
Recombinase Polymerase Amplification ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Molecular Testing ◽  
Analytical Sensitivity ◽  
Molecular Test ◽  
Current Gold Standard

AbstractThe COVID-19 pandemic has challenged testing capacity worldwide. The mass testing needed to stop the spread of the virus requires new molecular diagnostic tests that are faster and with reduced equipment requirement, but as sensitive as the current gold standard protocols based on polymerase chain reaction.We developed a fast (25-35 minutes) molecular test using reverse transcription recombinase polymerase amplification for simultaneous detection of two conserved regions of the virus, targeting the E and RdRP genes. The diagnostic platform offers two complementary detection methods: real-time fluorescence or visual dipstick.The analytical sensitivity of the test by real-time fluorescence was 9.5 (95% CI: 7.0-18) RNA copies per reaction for the E gene and 17 (95% CI: 11-93) RNA copies per reaction for the RdRP gene. The analytical sensitivity for the dipstick readout was 130 (95% CI: 82-500) RNA copies per reaction. The assay showed high specificity with both detection methods when tested against common seasonal coronaviruses, SARS-CoV and MERS-CoV model samples. The dipstick readout demonstrated potential for point-of-care testing, with simple or equipment-free incubation methods and a user-friendly prototype smartphone application was proposed with data capture and connectivity.This ultrasensitive molecular test offers valuable advantages with a swift time-to-result and it requires minimal laboratory equipment compared to current gold standard assays. These features render this diagnostic platform more suitable for decentralised molecular testing.

scholarly journals Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1731
Author(s):  
Arianna Ceruti ◽  
Rea Maja Kobialka ◽  
Judah Ssekitoleko ◽  
Julius Boniface Okuni ◽  
Sandra Blome ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Isolation ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Recombinase Polymerase Amplification ◽  
Reference Laboratory ◽  
Analytical Sensitivity ◽  
Lysis Buffer

African swine fever virus (ASFV) is the causative agent of a deadly disease in pigs and is spread rapidly across borders. Samples collected from suspected cases must be sent to the reference laboratory for diagnosis using polymerase chain reaction (PCR). In this study, we aimed to develop a simple DNA isolation step and real-time recombinase polymerase amplification (RPA) assay for rapid detection of ASFV. RPA assay based on the p72 encoding B646L gene of ASFV was established. The assays limit of detection and cross-reactivity were investigated. Diagnostic performance was examined using 73 blood and serum samples. Two extraction approaches were tested: silica-column-based extraction method and simple non-purification DNA isolation (lysis buffer and heating, 70 °C for 20 min). All results were compared with well-established real-time PCR. In a field deployment during a disease outbreak event in Uganda, 20 whole blood samples were tested. The assay’s analytical sensitivity was 3.5 DNA copies of molecular standard per µL as determined by probit analysis on eight independent assay runs. The ASFV RPA assay only detected ASFV genotypes. Compared to real-time PCR, RPA diagnostic sensitivity and specificity were 100%. Using the heating/lysis buffer extraction procedure, ASFV-RPA revealed better tolerance to inhibitors than real-time PCR (97% and 38% positivity rate, respectively). In Uganda, infected animals were identified before the appearance of fever. The ASFV-RPA assay is shown to be as sensitive and specific as real-time PCR. Moreover, the combination of the simple extraction protocol allows its use at the point of need to improve control measures.

scholarly journals Simultaneous Detection of North American and European Porcine Reproductive and Respiratory Syndrome Virus Using Real-Time Quantitative Reverse Transcriptase–PCR

2005 ◽  
Vol 17 (2) ◽  
pp. 165-170 ◽  
Author(s):  
Steven B. Kleiboeker ◽  
Susan K. Schommer ◽  
Sang-Myeong Lee ◽  
Sandy Watkins ◽  
Wayne Chittick ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
North American ◽  
Simultaneous Detection ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Field Isolates ◽  
Pcr Assays

Porcine reproductive and respiratory syndrome (PRRS) is 1 of the most economically important diseases of swine. Detection of the etiologic agent, PRRS virus (PRRSV), represents a diagnostic challenge due to the heterogeneity of field isolates as well as the propensity for swine to develop persistent infection in which virus is difficult to detect. Recently European (EU) lineage PRRSV isolates, which are genetically divergent from North American (NA) isolates, have been introduced into NA swine further complicating efforts to diagnose this disease. In this study, real-time ( TaqMan) reverse transcriptase (RT)–PCR assays were developed for multiplex detection, differentiation, and quantification of NA and EU PRRSV field isolates. Oligonucleotide primers and dual-labeled probes were selected from conserved regions of open-reading frame 7 and the 3'-untranslated region. The real-time RT-PCR assays described for the NA or EU genotype of PRRSV detected viral RNA from 83/83 strains (74 NA; 9 EU) previously isolated by cell culture between 1992 and 2003. The analytical sensitivity of both assays was consistently found to be less than a single TCID50, which corresponded to 5–10 RNA molecules, and was not significantly reduced when the reactions were performed in a multiplex format. When performing multiplex reactions, sensitive detection was possible even when 1 viral RNA concentration was up to 5,000-fold higher than the second. The diagnostic sensitivity and specificity of the multiplex reaction was found to be at a minimum equivalent to that of both nested RT-PCR and virus isolation.

scholarly journals Assessing a chip based rapid RTPCR test for SARS CoV-2 detection (TrueNat assay): A diagnostic accuracy study

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0257834
Author(s):  
Ujjala Ghoshal ◽  
Atul Garg ◽  
Shruthi Vasanth ◽  
Akshay K. Arya ◽  
Ankita Pandey ◽  
...  
Keyword(s):  
Diagnostic Accuracy ◽  
Real Time ◽  
Gold Standard ◽  
Standard Test ◽  
Molecular Diagnostic ◽  
Resource Limited ◽  
Accuracy Study ◽  
Trained Manpower ◽  
Sensitivity Specificity ◽  
Molecular Diagnostic Test

COVID-19 testing is required before admission of a patient in the hospitals, invasive procedures, major and minor surgeries etc. Real Time Polymerase chain reaction is the gold standard test for the diagnosis, but requires well equipped biosafety laboratory along with trained manpower. In this study we have evaluated the diagnostic accuracy of novel TrueNat molecular assay for detecting SARS CoV-2. TrueNat is a chip-based real time PCR test and works on portable, light weight, battery powered equipment and can be used in remote areas with poor infrastructure. In this study 1807 patients samples were collected for both TrueNat and RTPCR COVID-19 testing during study period. Of these 174 (9.7%) and 174 (15%) were positive by RTPCR and TrueNat respectively and taking results of RTPCR as gold standard TrueNat test showed a sensitivity, specificity and diagnostic accuracy of 69.5, 90.9% and 89.2% respectively. It can be concluded that TrueNat is a simple, easy to use, good rapid molecular diagnostic test for diagnosis of COVID-19 especially in resource limited settings and will prove to be a game changer of molecular diagnostics in future.

scholarly journals Rapid Detection and Differentiating of the Predominant Salmonella Serovars in Chicken Farm by TaqMan Multiplex Real-Time PCR Assay

2021 ◽  
Vol 11 ◽  
Author(s):  
Suhua Xin ◽  
Hong Zhu ◽  
Chenglin Tao ◽  
Beibei Zhang ◽  
Lan Yao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Epidemiologic Study ◽  
Clinical Diagnostics ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Chicken Farm

Salmonella has been known as an important zoonotic pathogen that can cause a variety of diseases in both animals and humans. Poultry are the main reservoir for the Salmonella serovars Salmonella Pullorum (S. Pullorum), Salmonella Gallinarum (S. Gallinarum), Salmonella Enteritidis (S. Enteritidis), and Salmonella Typhimurium (S. Typhimurium). The conventional serotyping methods for differentiating Salmonella serovars are complicated, time-consuming, laborious, and expensive; therefore, rapid and accurate molecular diagnostic methods are needed for effective detection and prevention of contamination. This study developed and evaluated a TaqMan multiplex real-time PCR assay for simultaneous detection and differentiation of the S. Pullorum, S. Gallinarum, S. Enteritidis, and S. Typhimurium. In results, the optimized multiplex real-time PCR assay was highly specific and reliable for all four target genes. The analytical sensitivity corresponded to three colony-forming units (CFUs) for these four Salmonella serovars, respectively. The detection limit for the multiplex real-time PCR assay in artificially contaminated samples was 500 CFU/g without enrichment, while 10 CFU/g after pre-enrichment. Moreover, the multiplex real-time PCR was applied to the poultry clinical samples, which achieved comparable results to the traditional bacteriological examination. Taken together, these results indicated that the optimized TaqMan multiplex real-time PCR assay will be a promising tool for clinical diagnostics and epidemiologic study of Salmonella in chicken farm and poultry products.

scholarly journals Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage

2003 ◽  
Vol 69 (9) ◽  
pp. 5178-5185 ◽  
Author(s):  
Rebecca A. Guy ◽  
Pierre Payment ◽  
Ulrich J. Krull ◽  
Paul A. Horgen
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Environmental Water ◽  
Qpcr Assay ◽  
Detection Methods ◽  
Environmental Water Samples ◽  
Dna Yield ◽  
Chelex 100

ABSTRACT The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the β-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

scholarly journals Utility of Nucleic Acid Extraction Free COVID-19 Real Time PCR Protocol in Resource Limited Setting: A Pilot Study

2021 ◽  
Author(s):  
Santosh Karade ◽  
Pratik Thosani ◽  
Prashant Patil ◽  
Kavita Bala Anand ◽  
Sourav Sen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Pilot Study ◽  
Real Time ◽  
Gold Standard ◽  
Rna Extraction ◽  
Molecular Diagnostic ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Resource Limited

Introduction: Coronavirus Disease (COVID-19), a respiratory infection, caused by severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, Hubei province, China in December 2019. Alarming increase in the number of cases has put tremendous pressure on existing health resources. Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), a molecular diagnostic method, is considered gold standard for diagnosis of SARS-CoV-2 infection. It involves RNA extraction as the preliminary step. Innovations to cut down cost and time involved in SARS-CoV-2 testing are need of hour. Aim: The aim of this study was to assess the feasibility of Nucleic Acid Extraction Free (NEF) protocol for COVID-19 diagnosis in resource limited settings. Materials and Methods: In this pilot study a panel of 148 Nasopharyngeal (NP) samples was subjected to the novel NEF RT-PCR protocol and results were compared to gold standard RT-PCR on RNA extracted from NP specimen. The cycle threshold value for each target was tabulated in MS Excel Spreadsheet and data analysis was performed using Statistical Package for Social Sciences (SPSS) software version 15.0. Results: Out of 148 collected samples, 120 showed amplification of E and RdRp targets by RNA extraction-based RT-PCR. Overall sensitivity and specificity observed for NEF protocol was 43.94% and 96.42%, respectively. Conclusion: Further refinement in the protocol would be required to improve the sensitivity of NEF protocol and widespread use in laboratories.

scholarly journals Rapid Detection of Enterocytozoon hepatopenaei Infection in Shrimp With a Real-Time Isothermal Recombinase Polymerase Amplification Assay

2021 ◽  
Vol 11 ◽  
Author(s):  
Chao Ma ◽  
Shihui Fan ◽  
Yu Wang ◽  
Haitao Yang ◽  
Yi Qiao ◽  
...  
Keyword(s):  
Real Time ◽  
Gel Electrophoresis ◽  
Rapid Detection ◽  
Bacteriophage T4 ◽  
Recombinase Polymerase Amplification ◽  
Detection Methods ◽  
Economic Losses ◽  
The Real ◽  
Enterocytozoon Hepatopenaei ◽  
Detection Speed

Enterocytozoon hepatopenaei (EHP) infection has become a significant threat in shrimp farming industry in recent years, causing major economic losses in Asian countries. As there are a lack of effective therapeutics, prevention of the infection with rapid and reliable pathogen detection methods is fundamental. Molecular detection methods based on polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have been developed, but improvements on detection speed and convenience are still in demand. The isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4 is promising, but the previously developed RPA assay for EHP detection read the signal by gel electrophoresis, which restricted this application to laboratory conditions and hampered the sensitivity. The present study combined fluorescence analysis with the RPA system and developed a real-time RPA assay for the detection of EHP. The detection procedure was completed in 3–7 min at 39°C and showed good specificity. The sensitivity of 13 gene copies per reaction was comparable to the current PCR- and LAMP-based methods, and was much improved than the RPA assay analyzed by gel electrophoresis. For real clinical samples, detection results of the real-time RPA assay were 100% consistent with the industrial standard nested PCR assay. Because of the rapid detection speed and the simple procedure, the real-time RPA assay developed in this study can be easily assembled as an efficient and reliable on-site detection tool to help control EHP infection in shrimp farms.

scholarly journals Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips

Biosensors ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 151
Author(s):  
Pei Wang ◽  
Lei Liao ◽  
Chao Ma ◽  
Xue Zhang ◽  
Junwei Yu ◽  
...  
Keyword(s):  
Food Safety ◽  
Vibrio Cholerae ◽  
Bacterial Infections ◽  
Vibrio Vulnificus ◽  
Simultaneous Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Detection Methods ◽  
Global Ocean ◽  
Clinical Samples

Vibrio cholerae and Vibrio vulnificus are two most reported foodborne Vibrio pathogens related to seafood. Due to global ocean warming and an increase in seafood consumption worldwide, foodborne illnesses related to infection of these two bacteria are growing, leading to food safety issues and economic consequences. Molecular detection methods targeting species-specific genes are effective tools in the fight against bacterial infections for food safety. In this study, a duplex detection biosensor based on isothermal recombinase polymerase amplification (RPA) and a three-segment lateral flow strip (LFS) has been established. The biosensor used lolB gene of Vibrio cholerae and empV gene of Vibrio vulnificus as the detection markers based on previous reports. A duplex RPA reaction for both targets were constructed, and two chemical labels, FITC and DIG, of the amplification products were carefully tested for effective and accurate visualization on the strip. The biosensor demonstrated good specificity and achieved a sensitivity of 101 copies per reaction or one colony forming unit (CFU)/10 g of spiked food for both bacteria. Validation with clinical samples showed results consistent with that of real-time polymerase chain reaction. The detection process was simple and fast with a 30-min reaction at 37 °C and visualization on the strip within 5 min. With little dependence on laboratory settings, this biosensor was suitable for on-site detection, and the duplex system enabled simultaneous detection of the two important foodborne bacteria. Moreover, the principle can be extended to healthcare and food safety applications for other pathogens.

scholarly journals Evaluation of a fully automated high-throughput SARS-CoV-2 multiplex qPCR assay with build-in screening functionality for DelHV69/70- and N501Y variants such as B.1.1.7

2021 ◽  
Author(s):  
Dominik Nörz ◽  
Moritz Grunwald ◽  
Flaminia Olearo ◽  
Nicole Fischer ◽  
Martin Aepfelbacher ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Simultaneous Detection ◽  
Multiplex Assay ◽  
Detection Methods ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Highly Sensitive

1AbstractBackgroundNew SARS-CoV-2 variants with increased transmissibility, like B.1.1.7 from England or B1.351 from South Africa, have caused considerable concern worldwide. In order to contain the spread of these lineages, it is of utmost importance to have rapid, sensitive and high-throughput detection methods at hand.MethodsAnalytical sensitivity was assessed for both wild-type SARS-CoV-2 and B.1.1.7 lineage by serial dilution. A total of 141 clinical samples were subjected to the test and results compared to a commercial manual typing-PCR assay and NGS.ResultsThe multiplex assay is highly sensitive for detection of SARS-CoV-2 RNA in clinical samples, with an LoD of 25.82 cp/ml (CI: 11.61 – 57.48). LoDs are slightly higher for the HV68/70 deletion (111.36 cp/ml; CI: 78.16 – 158.67) and the N501Y SNP (2548.04 cp/ml, CI: 1592.58 – 4076.73). A total of 141 clinical samples were tested with the assay, including 16 samples containing SARS-CoV-2 of the B.1.1.7 lineage. Three non-B.1.1.7 samples contained a HV69/70 deletion. All were correctly identified by the multiplex assay.ConclusionWe describe here a highly sensitive, fully automated multiplex PCR assay for the simultaneous detection of del-HV69/70 and N501Y that can distinguish between lineages B.1.1.7 and B1.351. The assay allows for high-throughput screening for relevant variants in clinical samples prior to sequencing.

Identification of real-time PCR-negative EGFR mutations by direct sequencing test.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22118-e22118
Author(s):  
Tao-Yeuan Wang ◽  
Chi-Kuan Chen
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Egfr Mutation ◽  
Detection Efficiency ◽  
Direct Sequencing ◽  
Egfr Mutations ◽  
Molecular Diagnostic ◽  
Molecular Testing ◽  
Mutation Site ◽  
Pcr Test

e22118 Background: High percentage of patients with non-small cell lung cancer (NSCLC) has EGFR activating mutations in Taiwan and responses to EGFR inhibitors such as Tarceva (1, 2, 3). Timely molecular diagnosis is critical and molecular testing should be performed before treatment for the individual patient. It is important to develop an effective molecular diagnostic method for accurately detecting mutations and preventing false positive results. Therefore, we used a universal genetic detecting method (FemtoPath) to increase sensitivity of gene mutation detection and improve the sensitivity limitation (0.1%). Our previous reports indicated that FemtoPath/direct sequencing test is more sensitive, accurate and inexpensive than Cobas KRAS real- time PCR mutation test. Methods: In this study, we compared two molecular testing methods for the detection of EGFR mutation; the direct sequencing and the real-time PCR. Results: FemtoPath EGFR direct sequencing test identified mutations in 57 of the 73 tumors (77.03%) and Cobas EGFR mutation test identifies mutations in 53 of the 73 tumors (72.97%). Six negative results obtained by Real-Time PCR test were re-confirmed to be positive by direct sequencing test. From the results above, FemtoPath/direct sequencing test not only accurately detected all of the mutations, but also identified mutations adjacent toexons 18, 19, 20 and 21 of the EGFR gene. In 52 consensus cases of EGFR mutation obtained from both methods, there were 10 major discrepancies including two S768, four Exon 19 deletion, one L858, five T790, and one exon 20 insertion in this experiment. Conclusions: FemtoPath/direct sequencing test has lower invalid rate than Real-Time PCR test while fewer sample amount was required with increased detection efficiency of unknown mutation site.

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