scholarly journals Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Xin-na Li ◽  
Xin-xin Shen ◽  
Ming-hui Li ◽  
Ju-ju Qi ◽  
Rui-huan Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Enterovirus 71 ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Coxsackievirus A16 ◽  
Fluorescence Detector ◽  
Lateral Flow Strip ◽  
Clinical Diagnostic

Abstract Background Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. Methods Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. Results The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. Conclusions The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

scholarly journals Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities

Biosensors ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 11
Author(s):  
Zhijian Yi ◽  
Jean de Dieu Habimana ◽  
Omar Mukama ◽  
Zhiyuan Li ◽  
Nelson Odiwuor ◽  
...  
Keyword(s):  
Real Time ◽  
Early Stage ◽  
Limited Resource ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Capture Probe ◽  
Fluorescence Detector ◽  
Lateral Flow Strip ◽  
Global Pandemic ◽  
Novel Approach

Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.

scholarly journals Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae

2020 ◽  
Author(s):  
Jinfeng Wang ◽  
Ruiwen Li ◽  
Xiaoxia Sun ◽  
Libing Liu ◽  
Xuepiao Hao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Metal Bath ◽  
Lateral Flow Strip ◽  
The Real ◽  
Mycoplasmal Pneumonia ◽  
Mycoplasma Ovipneumoniae

Abstract Background: Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae is one of major etiological agent causing mycoplasmal pneumonia. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens. Results: The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. ovipneumoniae targeting a conserved region of the 16S rRNA gene. Real-time RPA was performed in a portable florescence scanner at 39 °C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39 °C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for M. ovipneumoniae , as there were no cross-reactions with other microorganisms tested, especially the pathogens involved in respiratory complex and other mycoplasmas frequently identified in ruminant s . The limit of detection of LFS RPA assay was 1.0×10 1 copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times lower than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 111 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 29 samples in the real-time RPA, 31 samples in the LFS RPA and 32 samples in the real-time PCR assay. Compared to real-time PCR, the real-time RPA and LFS RPA showed diagnostic specificity of 100% and 98.73%, diagnostic sensitivity of 90.63% and 93.75%, and a kappa coefficient of 0.932 and 0.934, respectively. Conclusions: The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of M. ovipneumoniae , especially in resource-limited settings.

scholarly journals Specific Detection of Influenza A and B Viruses by CRISPR-Cas12a-Based Assay

Biosensors ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 88
Author(s):  
Bum Ju Park ◽  
Man Seong Park ◽  
Jae Myun Lee ◽  
Yoon Jae Song
Keyword(s):  
Diagnostic Test ◽  
Reverse Transcription ◽  
Influenza A ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Influenza Viruses ◽  
Nucleic Acid Amplification ◽  
Lateral Flow Strip ◽  
Influenza Pandemics

A rapid and accurate on-site diagnostic test for pathogens including influenza viruses is critical for preventing the spread of infectious diseases. Two types of influenza virus, A and B cause seasonal flu epidemics, whereas type A can cause influenza pandemics. To specifically detect influenza A (IAV) and B (IBV) viruses, we developed a clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system-based assay. By coupling reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), a CRISPR-Cas12a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) detected IAV and IBV titers as low as 1 × 100 plaque forming units (PFUs) per reaction without exhibiting cross-reactivity. Only 75 to 85 min were required to detect IAV and IBV, depending on isothermal nucleic acid amplification methods, and results were verified using a lateral flow strip assay that does not require additional analytic equipment. Taken together, our findings establish RT-RPA and RT-LAMP-coupled DETECTR-based diagnostic tests for rapid, specific and high-sensitivity detection of IAV and IBV using fluorescence and lateral flow assays. The diagnostic test developed in this study can be used to distinguish IAV and IBV infections, a capability that is necessary for monitoring and preventing the spread of influenza epidemics and pandemics.

scholarly journals A Novel Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification Detection Platform: Application to Diagnosis of COVID-19

2021 ◽  
Vol 9 ◽  
Author(s):  
Yi Wang ◽  
Xiaoxia Wang ◽  
Hai Chen ◽  
Limei Han ◽  
Licheng Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Virus Disease ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Health Concern ◽  
One Pot ◽  
Loop Mediated Isothermal Amplification

The ongoing Corona virus disease (COVID-19) outbreak has become a huge global health concern. Here, we reported a novel detection platform based on the loop-mediated isothermal amplification (LAMP), termed real-time reverse transcription LAMP (rRT-LAMP) and applied it for the diagnosis of COVID-19 (COVID-19 rRT-LAMP). rRT-LAMP integrates reverse transcription, LAMP amplification, restriction endonuclease cleavage and real-time fluorescence detection into one-pot reaction, and facilitates the diagnosis of COVID-19 at 64°C for only 35 min. The ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2 were detected for diagnosing COVID-19. The limit of detection (LoD) of COVID-19 rRT-LAMP assay was 14 copies (for each marker) per vessel, and no positive results were obtained from non-SARS-CoV-2 templates. To demonstrate its feasibility, a total of 33 oropharynx swab samples collected from COVID-19 patients also were diagnosed as SARS-CoV-2 infection using COVID-19 rRT-LAMP protocol. No cross-reactivity was yielded from 41 oropharynx swab samples collected from non-COVID-19 patients. These data suggesting that the COVID-19 rRT-LAMP assay is a potential detection tool for the diagnosis of SARS-CoV-2 infection in clinical, field and disease control laboratories, and will be valuable for controlling the COVID-19 epidemic.

scholarly journals Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae

2020 ◽  
Author(s):  
Jinfeng Wang ◽  
Ruiwen Li ◽  
Xiaoxia Sun ◽  
Libing Liu ◽  
Xuepiao Hao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Metal Bath ◽  
Lateral Flow Strip ◽  
The Real ◽  
Mycoplasmal Pneumonia ◽  
Mycoplasma Ovipneumoniae

Abstract Background Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae is one of major etiological agent causing mycoplasmal pneumonia. It is an urgent need to develop a rapid and accurate method to detect M. ovipneumoniae . Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens. Results The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. ovipneumoniae targeting a conserved region of the 16SrRNA gene. Real-time RPA was performed in a portable florescence scanner at 39 °C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39 °C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for M. ovipneumoniae , as there were no cross-reactions with other pathogens tested, especially the M. capricolum subsp. capripneumoniae . The limit of detection of LFS RPA assay was 1.0×10 1 copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times higher than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 46 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 17 samples in the RPA assays and 19 samples in the real-time PCR assay. The real-time RPA and LFS RPA showed diagnostic specificity of 100%, diagnostic sensitivity of 89.47%, and a kappa coefficient of 0.909. Conclusions The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of M. ovipneumoniae , especially in resource-limited settings.

Development of a real-time reverse transcription-PCR assay to detect and quantify group A rotavirus equine-like G3 strains

2021 ◽  
Author(s):  
Eric M. Katz ◽  
Mathew D. Esona ◽  
Rashi Gautam ◽  
Michael D. Bowen
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Reverse Transcription ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Human Populations ◽  
Pcr Assay ◽  
Group A Rotavirus ◽  
Reverse Transcription Pcr ◽  
Group A

Since 2013, group A rotavirus strains characterized as novel DS-1-like inter-genogroup reassortant ‘equine-like G3’ strains have emerged and spread across five continents among human populations in at least 14 countries. Here we report a novel one-step TaqMan quantitative real-time reverse transcription-PCR assay developed to genotype and quantify the viral load for samples containing rotavirus equine-like G3 strains. Using a universal G forward primer and a newly designed reverse primer and TaqMan probe, we developed and validated an assay with a linear dynamic range of 2.3 × 10 9 – 227 copies per reaction and a limit of detection of 227 copies. The percent positive agreement, percent negative agreement, and precision of our assay were 100.00%, 99.63%, and 100.00%, respectively. This assay can simultaneously detect and quantify the viral load for samples containing DS-1-like inter-genogroup reassortant equine-like G3 strains with high sensitivity and specificity, faster turnaround time, and decreased cost and will be valuable for high-throughput screening of stool samples collected to monitor equine-like G3 strain prevalence and circulation among human populations throughout the world.

scholarly journals VP2 Dominated CD4+T Cell Responses against Enterovirus 71 and Cross-Reactivity against Coxsackievirus A16 and Polioviruses in a Healthy Population

2013 ◽  
Vol 191 (4) ◽  
pp. 1637-1647 ◽  
Author(s):  
Shuguang Tan ◽  
Xiaojuan Tan ◽  
Xiaoman Sun ◽  
Guangwen Lu ◽  
Chun-Chi Chen ◽  
...  
Keyword(s):  
T Cell ◽  
Enterovirus 71 ◽  
T Cell Responses ◽  
Cross Reactivity ◽  
Cd4 T Cell ◽  
Healthy Population ◽  
Coxsackievirus A16 ◽  
Cell Responses

scholarly journals Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis

2005 ◽  
Vol 109 (4) ◽  
pp. 365-379 ◽  
Author(s):  
Stephen A. Bustin ◽  
Reinhold Mueller
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Therapy Monitoring ◽  
Clinical Diagnostics ◽  
Clinical Diagnostic Laboratory ◽  
Diagnostic Laboratory ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Clinical Diagnostic ◽  
Pcr Assays

qRT-PCR (real-time reverse transcription-PCR) has become the benchmark for the detection and quantification of RNA targets and is being utilized increasingly in novel clinical diagnostic assays. Quantitative results obtained by this technology are not only more informative than qualitative data, but simplify assay standardization and quality management. qRT-PCR assays are most established for the detection of viral load and therapy monitoring, and the development of SARS (severe acute respiratory syndrome)-associated coronavirus qRT-PCR assays provide a textbook example of the value of this technology for clinical diagnostics. The widespread use of qRT-PCR assays for diagnosis and the detection of disease-specific prognostic markers in leukaemia patients provide further examples of their usefulness. Their value for the detection of disease-associated mRNA expressed by circulating tumour cells in patients with solid malignancies is far less apparent, and the clinical significance of results obtained from such tests remains unclear. This is because of conceptual reservations as well as technical limitations that can interfere with the diagnostic specificity of qRT-PCR assays. Therefore, although it is evident that qRT-PCR assay has become a useful and important technology in the clinical diagnostic laboratory, it must be used appropriately and it is essential to be aware of its limitations if it is to fulfil its potential.

scholarly journals Multicenter Clinical Validation of a Cartridge-Based Real-Time PCR System for Detection of Coccidioides spp. in Lower Respiratory Specimens

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
Michael A. Saubolle ◽  
Bette R. Wojack ◽  
Anne M. Wertheimer ◽  
Atehkeng Z. Fuayagem ◽  
Stephen Young ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Clinical Test ◽  
Target Sequence ◽  
Fungal Culture ◽  
Content Type ◽  
Bronchial Wash

ABSTRACT Available methods for the diagnosis of coccidioidomycosis have significant shortcomings relative to accuracy and timeliness. We retrospectively and prospectively evaluated the diagnostic performance and reproducibility of a new cartridge-based real-time PCR assay for Coccidioides spp. directly in lower respiratory secretions and compared them to today's “gold standard,” fungal culture. The GeneSTAT Coccidioides assay uses a 106-bp target sequence repeated multiple times (∼60×) per genome, thus lowering the limit of detection (LOD) for extracted DNA to 10 genome equivalents/ml. A total of 332 prospective and retrospective individual patient specimens were tested. The retrospective samples consisted of 100 bronchoalveolar lavage or bronchial wash (BAL/BW) (51 positive and 49 negative by culture) specimens that had been collected previously and stored at −70°C. These samples were tested by the GeneSTAT Coccidioides assay across three clinical test sites. The sensitivity was 100%, and the specificity ranged between 93.8% and 100%. There was minimal variance in the percent agreement across the three sites, 95.6% to 100%. Additionally, a total of 232 fresh (prospective) deidentified BAL/BW specimens were tested across the three clinical sites, which included a number of specimens from Southern California to provide a diversity of isolates. Specimens were tested by fungal culture, with any isolates of Coccidioides, except for one, being confirmed by molecular means (AccuProbe). The sensitivity of the GeneSTAT Coccidioides assay across the three sites was 100% (4/4) for positive fresh specimens, and the overall specificity of the assay was 99.6% (227/228), ranging from 98.1% to 100%. In testing for cross-reactivity, the assay was 100% specific when screened against 47 different bacterial, viral, and fungal species.

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