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 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.

scholarly journals Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246573
Author(s):  
Sandeep K. Gupta ◽  
Qing Deng ◽  
Tanushree B. Gupta ◽  
Paul Maclean ◽  
Joerg Jores ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Clinical Samples ◽  
The Real ◽  
Positive Rate ◽  
Mycoplasma Ovipneumoniae

Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

scholarly journals Direct and Rapid Detection of Mycoplasma bovis in Bovine Milk Samples by Recombinase Polymerase Amplification Assays

2021 ◽  
Vol 11 ◽  
Author(s):  
Ruiwen Li ◽  
Jinfeng Wang ◽  
Xiaoxia Sun ◽  
Libing Liu ◽  
Jianchang Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Bovine Milk ◽  
Bovine Mastitis ◽  
High Specificity ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Mycoplasma Bovis ◽  
Lateral Flow Strip ◽  
The Real ◽  
Milk Samples

This study aimed to detetct Mycoplasma bovis (M. bovis) in bovine milk quickly and directly by developing and validating isothermal recombinase polymerase amplification (RPA) assays. Targeting the uvrC gene of M. bovis, an RPA assay based on the fluorescence monitoring (real-time RPA) and an RPA assay combined with a lateral flow strip (LFS RPA) were conducted. It took 20 min for the real-time RPA to finish in a Genie III at 39°C, and 15 min were required to perform the LFS RPA in an incubator block at 39°C, followed by the visualization of the products on the lateral flow strip within 5 min. Both of the two assays showed high specificity for M. bovis without any cross-reaction with the other tested pathogens. With the standard recombinant plasmid pMbovis-uvrC serving as a template, both RPA assays had a limit of detcion of 1.0 × 101 copies per reaction, equivalent to that of a real-time PCR assay. In the 65 milk samples collected from cattle with mastitis, the M. bovis genomic DNA was detected in 24 samples by both the real-time RPA and the LFS RPA assays. The developed RPA assays could detect M. bovis in bovine milk in an efficient, convenient, and credible manner as attractive and promising tools, and the assays would be helpful in the rapid response to M. bovis infection causing bovine mastitis.

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 Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of Salmonella spp. in Food Samples

2021 ◽  
Vol 11 ◽  
Author(s):  
Liwei Zhao ◽  
Jianchang Wang ◽  
Xiao Xia Sun ◽  
Jinfeng Wang ◽  
Zhimin Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Protein A ◽  
Food Samples ◽  
Cross Reactivity ◽  
Salmonella Spp ◽  
Lateral Flow Strip ◽  
The Real ◽  
Conserved Sequence

Salmonella spp. is among the main foodborne pathogens which cause serious foodborne diseases. An isothermal real-time recombinase polymerase amplification (RPA) and lateral flow strip detection (LFS RPA) were used to detect Salmonella spp. targeting the conserved sequence of invasion protein A (invA). The Real-time RPA was performed in a portable florescence scanner at 39°C for 20 min. The LFS RPA was performed in an incubator block at 39°C for 15 min, under the same condition that the amplifications could be inspected by the naked eyes on the LFS within 5 min. The detection limit of Salmonella spp. DNA using real-time RPA was 1.1 × 101 fg, which was the same with real-time PCR but 10 times higher than that of LFS RPA assay. Moreover, the practicality of discovering Salmonella spp. was validated with artificially contaminated lamb, chicken, and broccoli samples. The analyzing time dropped from 60 min to proximately 5–12 min on the basis of the real-time and LFS RPA assays compared with the real-time PCR assay. Real-time and LFS RPA assays’ results were equally reliable. There was no cross-reactivity with other pathogens in both assays. In addition, the assays had good stability. All of these helped to show that the developed RPA assays were simple, rapid, sensitive, credible, and could be a potential point-of-need (PON) test required mere resources.

Real-Time PCR Assay for Detection of Kingella kingae in Children

2018 ◽  
Vol 13 (03) ◽  
pp. 216-223
Author(s):  
Theresa Madigan ◽  
Scott Cunningham ◽  
Poornima Ramanan ◽  
Micah Bhatti ◽  
Robin Patel
Keyword(s):  
Synovial Fluid ◽  
Septic Arthritis ◽  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Toxin Gene ◽  
Pcr Assay ◽  
Kingella Kingae ◽  
The Real ◽  
Osteoarticular Infections

Background Kingella kingae is a known cause of osteoarticular infections in children younger than 4 years of age, but it is not always recoverable in culture. Molecular methods are increasingly used for diagnosis. Methods To facilitate diagnosis of K. kingae septic arthritis, we developed a real-time polymerase chain reaction (PCR) assay for the detection of K. kingae that targets the repeat-in-toxin gene (rtxB). Results We present three pediatric patients with K. kingae septic arthritis at our institution who were diagnosed using the real-time PCR assay. All underwent arthrotomy with irrigation and debridement and were symptom-free after 3 weeks of therapy with β-lactam antibiotics. Cultures of synovial fluid or tissue grew K. kingae in two of three; K. kingae real-time PCR was positive in all three patients. In addition, 11 cases of K. kingae osteoarticular infection were diagnosed through Mayo Medical Laboratories using this assay. The limit of detection of the real-time PCR assay was 73.7 colony-forming unit (CFU)/µL for tissue and 1.3 CFU/µL for synovial fluid. Conclusions PCR-based detection methods are faster and more sensitive than conventional culture-based methods for the diagnosis of K. kingae osteoarticular infections in children.

scholarly journals Rapid Monitoring of Vancomycin Concentration in Serum Using Europium (III) Chelate Nanoparticle-Based Lateral Flow Immunoassay

2021 ◽  
Vol 9 ◽  
Author(s):  
Lun Bian ◽  
Junyu Liang ◽  
Hui Zhao ◽  
Ke Ye ◽  
Zhaoyue Li ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Lateral Flow Immunoassay ◽  
Real Time Monitoring ◽  
Competitive Immunoassay ◽  
Vancomycin Concentration ◽  
The Real ◽  
Coefficients Of Variation ◽  
Rapid Monitoring

Establishing personalized medication plans for patients to maximize therapeutic efficacy and minimize the toxicity of vancomycin (VAN) requires rapid, simple, and accurate monitoring of VAN concentration in body fluid. In this study, we have developed a simple and rapid analytical method by integrating Eu (III) chelate nanoparticles (CN-EUs) and lateral flow immunoassay (LFIA) to achieve the real-time monitoring of VAN concentration in serum within 15 min. This approach was performed on nitrocellulose (NC) membrane assembled LFIA strips via indirect competitive immunoassay and exhibited a wide linear range of detection (0.1–80 μg*ml−1) with a low limit of detection (69.2 ng*ml−1). The coefficients of variation (CV) of the intra- and inter-assay in the detection of VAN were 7.12–8.53% and 8.46–11.82%, respectively. The dilution test and specificity indicated this method had a stability that was not affected by the serum matrix and some other antibiotics. Furthermore, the applicability of the proposed method was assessed by comparing the determined results with those measured by LC-MS/MS, showing a satisfactory correlation (R2 = 0.9713). The proposed CN-EUs-based LFIA manifested promising analytical performance, which showed potential value in the real-time monitoring of VAN and could help optimize the clinical use of more antibiotics.

scholarly journals Rapid detection of extended spectrum β-lactamase producing Escherichia coli isolated from fresh pork meat and pig cecum samples using multiplex recombinase polymerase amplification and lateral flow strip analysis

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248536
Author(s):  
Sitthichai Kanokudom ◽  
Thachaporn Assawakongkarat ◽  
Yukihiro Akeda ◽  
Panan Ratthawongjirakul ◽  
Rungtip Chuanchuen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Nucleotide Sequencing ◽  
Pork Meat ◽  
Lateral Flow Strip ◽  
Extended Spectrum

The emergence and dissemination of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is a global health issue. Food-producing animals, including pigs, are significant reservoirs of antimicrobial resistance (AMR), which can be transmitted to humans. Thus, the rapid detection of ESBLs is required for efficient epidemiological control and treatment. In this study, multiplex recombinase polymerase amplification (RPA) combined with a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS), as a lateral flow strip assay (LFA), was established for the rapid and simultaneous detection of multiple bla genes in a single reaction. Visible blue lines, indicating the presence of the blaCTX-M, blaSHV, and blaOXA genes, were observed within 10 min by the naked eye. The limit of detection of all three genes was 2.5 ng/25 μL, and no cross-reactivity with seven commensal aerobic bacteria was observed. A total of 93.9% (92/98) and 96% (48/50) of the E. coli isolates from pork meat and fecal samples, respectively, expressed an ESBL-producing phenotype. Nucleotide sequencing of the PCR amplicons showed that blaCTX-M was the most prevalent type (91.3–95.83%), of which the main form was blaCTX-M-55. The sensitivity and specificity of the RPA-LFA were 99.2% and 100%, respectively, and were in almost perfect agreement (κ = 0.949–1.000) with the results from PCR sequencing. Thus, the RPA-LFA is a promising tool for rapid and equipment-free ESBL detection and may facilitate clinical diagnosis in human and veterinary medicine, as well as AMR monitoring and surveillance.

APPLICATION OF REAL-TIME PRC TECHNIQUE AND REVERSE DOT-BLOT FOR DETECTION THE HIGH RISK TYPES OF HUMAN PAPILLOMAVIRUS CAUSING CERVICAL CANCER

2017 ◽  
pp. 99-103
Author(s):  
Van Bao Thang Phan ◽  
Hoang Bach Nguyen ◽  
Van Thanh Nguyen ◽  
Thi Nhu Hoa Tran ◽  
Viet Quynh Tram Ngo
Keyword(s):  
Cervical Cancer ◽  
High Risk ◽  
Real Time ◽  
Real Time Pcr ◽  
Dot Blot ◽  
The Real ◽  
Dot Blot Assay ◽  
Hpv Types ◽  
High Risk Hpv ◽  
Reverse Dot Blot

Introduction: Infection with HPV is the main cause of cervical cancer. Determining HPV infection and the types of HPV plays an important role in diagnosis, treatment and prognosis of cervicitis/cervical cancer. Aims: Determining proportion of high-risk HPV types and the occurrence of coinfection with multiple HPV types. Methods: 177 women with cervicitis or abnormal Pap smear result were enrolled in the study. Performing the real-time PCR for detecting HPV and the reverse DOT-BLOT assay for determining type of HPV in cases of positive PCR. Results: 7 types of high-risk HPV was dectected, the majority of these types were HPV type 18 (74.6%) and HPV type 16 (37.6%); the proportion of infection with only one type of HPV was 30.4% and coinfection with multiple HPV types was higher (69.6%), the coinfected cases with 2 and 3 types were dominated (32.2% and 20.3%, respectively) and the coinfected cases with 4 and 5 types were rare. Conclusion: Use of the real-time PCR and reverse DOT-BLOT assay can determine the high-risk HPV types and the occurrence of coinfection with multiple HPV types. Key words: HPV type, Reverse DOT-BLOT, real-time PCR,PCR, cervical cancer

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