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

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.

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

10.21203/rs.3.rs-17865/v2 ◽

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.

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

10.21203/rs.3.rs-17865/v1 ◽

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.

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Development of a Real-Time PCR for Detection of Mycoplasma Bovis in Bovine Milk and Lung Samples

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870501700603 ◽

2005 ◽

Vol 17 (6) ◽

pp. 537-545 ◽

Cited By ~ 47

Author(s):

Hugh Y. Cai ◽

Patricia Bell-Rogers ◽

Lois Parker ◽

John F. Prescott

Keyword(s):

Real Time ◽

Lung Tissue ◽

Real Time Pcr ◽

Bovine Milk ◽

Melting Peak ◽

Clinical Samples ◽

Mycoplasma Bovis ◽

Pcr Assay ◽

Milk Samples ◽

Culture Positive

A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6°C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1°C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 × 104 and 7.7 × 108 cfu/ml and the M. bovis culture-positive lungs between 1 × 103 and 1 × 109 cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.

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Development of real-time and lateral flow strip reverse transcription recombinase polymerase Amplification assays for rapid detection of peste des petits ruminants virus

Virology Journal ◽

10.1186/s12985-017-0688-6 ◽

2017 ◽

Vol 14 (1) ◽

Cited By ~ 23

Author(s):

Yang Yang ◽

Xiaodong Qin ◽

Yiming Song ◽

Wei Zhang ◽

Gaowei Hu ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Rapid Detection ◽

Lateral Flow ◽

Recombinase Polymerase Amplification ◽

Peste Des Petits Ruminants ◽

Lateral Flow Strip

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Rapid Detection of Mycoplasma bovis, Staphylococcus aureus and Streptococcus agalactiae in Cattle Bulk Tank Milk in Cyprus and Relations with Somatic Cell Counts

Pathogens ◽

10.3390/pathogens10070841 ◽

2021 ◽

Vol 10 (7) ◽

pp. 841

Author(s):

Maria Liapi ◽

George Botsaris ◽

Costas Arsenoglou ◽

Nikolas Markantonis ◽

Christodoulos Michael ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Somatic Cell ◽

Real Time ◽

Streptococcus Agalactiae ◽

Mycoplasma Bovis ◽

Bulk Tank Milk ◽

Somatic Cell Counts ◽

Milk Samples ◽

Cell Counts ◽

Bulk Tank

One hundred and seventy-seven (177) bulk tank milk samples were analyzed with a commercially available real-time polymerase chain reaction kit and 11 (6.21%), 41 (23.16%), and 58 (32.77%) tested positive for Mycoplasma bovis, Staphylococcus aureus, and Streptococcus agalactiae, respectively. Statistical analysis revealed a significant relationship between the presence of S. aureus and S. agalactiae. Enumeration of somatic cells was performed in the same samples by flow cytometry. The somatic cell counts were found higher in S. aureus and S. agalactiae positive samples. No association was found between M. bovis presence and somatic cells counts. Low internal assay control Ct values were found to be related with high somatic cell counts. Noticeably, this is the first report for the presence of M. bovis in Cyprus. Therefore, its presence was confirmed by bulk tank milk culture, conventional PCR, and next generation sequencing. Furthermore, M. bovis was typed with multilocus sequencing typing and was allocated to sequence type 29 (ST 29). Real-time PCR in bulk tank milk samples is a useful tool to detect mammary infections, especially for neglected pathogens such as M. bovis.

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Mycoplasmal mastitis in dairy cows in the Moghan region of Ardabil State, Iran : short communication

Journal of the South African Veterinary Association ◽

10.4102/jsava.v77i4.382 ◽

2006 ◽

Vol 77 (4) ◽

Cited By ~ 5

Author(s):

C. Ghazaei

Keyword(s):

Dairy Cows ◽

Short Communication ◽

Bovine Mastitis ◽

Subclinical Mastitis ◽

Clinical Mastitis ◽

Mycoplasma Bovis ◽

Bulk Tank Milk ◽

Milk Samples ◽

The World ◽

Bulk Tank

Mycoplasmas are an important and economically significant cause of mastitis in dairy cows in various parts of the world. The organisms are highly contagious, with the main reservoir of infection originating from cows with subclinical mastitis. In 1998 the 1st cases of bovine mastitis due to Mycoplasma bovis were diagnosed in Ardabil State, Iran. An investigation was carried out with the aim of establishing the extent of mycoplasma infections in dairy cows in Ardabil State. Milk samples obtained from 80 cows with clinical mastitis were cultured in the laboratory for the presence of mycoplasmas. Similarly, 48 bulk-tank milk samples were examined for the presence of mycoplasmas. A modified Hayflick broth was used to isolate the mycoplasmas and an immunoperoxidase test used for the species identification of the isolates. Mycoplasma bovis was isolated from 39 (48.75 %) of the clinical mastitis samples and from 48 of the bulk-tank milk samples tested. This indicated that mycoplasma udder infections were more prevalent in dairy cows in Ardabil State than previously thought.

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

Biosensors ◽

10.3390/bios12010011 ◽

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.

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Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus

Virology Journal ◽

10.1186/s12985-017-0792-7 ◽

2017 ◽

Vol 14 (1) ◽

Cited By ~ 11

Author(s):

Yang Yang ◽

Xiaodong Qin ◽

Xiangle Zhang ◽

Zhixun Zhao ◽

Wei Zhang ◽

...

Keyword(s):

Real Time ◽

Rapid Detection ◽

Lateral Flow ◽

Recombinase Polymerase Amplification ◽

Sheeppox Virus ◽

Goatpox Virus ◽

Lateral Flow Dipstick

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Development of recombinase polymerase amplification combined with lateral flow detection assay for rapid and visual detection of Ralstonia solanacearum in tobacco

Plant Disease ◽

10.1094/pdis-04-21-0688-re ◽

2021 ◽

Author(s):

Changfeng Li ◽

Yuliang Ju ◽

Xun Wu ◽

Pengfei Shen ◽

Le Cao ◽

...

Keyword(s):

Ralstonia Solanacearum ◽

Visual Detection ◽

High Sensitivity ◽

High Specificity ◽

Lateral Flow ◽

Recombinase Polymerase Amplification ◽

Molecular Diagnostic ◽

Tobacco Stem ◽

Detection Assay ◽

Flow Detection

Bacterial wilt caused by Ralstonia solanacearum is a serious soil-borne disease that results in severe losses to tobacco (Nicotiana tabacum) production in China. In this study, a novel RPA-LFD assay for the rapid visual detection of R. solanacearum was established using recombinase polymerase amplification (RPA) and lateral-flow dipstick (LFD). The RPA-LFD assay was performed at 37°C in 30 min without complex equipment. Targeting the sequence of the RipTALI-9 gene, we designed RPA primers (Rs-rpa-F/R) and an LF probe (Rs-LF-probe) that showed high specificity to R. solanacearum. The sensitivity of RPA-LFD assay to R. solanacearum was the same as that in conventional PCR at 1 pg genomic DNA, 102 CFU/g artificially inoculated tobacco stem, and 103 CFU/g artificially inoculated soil. The RPA-LFD assay could also detect R. solanacearum from plant and soil samples collected from naturally infested tobacco fields. These results suggest that the RPA-LFD assay developed in this study is a rapid, accurate molecular diagnostic tool with high sensitivity for the detection of R. solanacearum.

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