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Modified loop-mediated isothermal amplification method combined with a TaqMan probe for the detection of Mycoplasma gallisepticum

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Abstract

We established and validated an improved loop-mediated isothermal amplification (LAMP) method combined with a TaqMan probe (LAMp-Taqman method) for detection of Mycoplasma gallinis (MG). A test plasmid was constructed using a conserved sequence of the specific gene vlhA of MG, and the LAMP primer set was screened according to the conservative gene design. The loop primer modified by fluorescent/quenching dyes was used as the TaqMan probe to establish the LAMP-TaqMan detection method. The feasibility and application value of LAMP in detecting MG nucleic acids were evaluated by parallel testing using real-time fluorescence quantitative qPCR as a control. The study results showed that LAMP-TaqMan had good specificity in detecting MG nucleic acids. The optimal detection temperature was 63 ºC, and the linear correlation coefficient R2 of eight tenfold serial dilutions (2.607 × 106 copies/µL–2.607 × 10−1 copies/µL) was 0.995; the minimum detection limit was 2.607–1 copies/µL, and the detection rate of the minimum detection limit was 100%. The high-, medium-, and low-concentration test plasmids were tested for repeatability (the coefficients of variation (CVs) were 0.202%, 1.770%, and 3.771%, respectively). Parallel tests were conducted on 120 samples collected from pairs of chickens, the accuracy, sensitivity and specificity of TP-LAMP were 97.50%, 100.00% and 96.30%, respectively. The detection time is 1 h, in fact, test results can be obtained in 45 min. The LAMP-TaqMan isothermal nucleic acid detection method not only maintains the effect of the loop primer to accelerate amplification but also offers fast and simple detection, a low detection limit, and a low rate of false positives; it has a broad market application prospect in the accurate and rapid detection of poultry diseases.

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Data availability

The datasets generated during the current study are available from the corresponding author upon reasonable request.

References

  1. Hakeem W, Farran MT, Shaib HA et al (2021) Does methionine enhance immunity in Mycoplasma gallispeticum F strain vaccinated broiler breeder pullets? J Appl Poult Res 30(3):100172

    Article  Google Scholar 

  2. Javed MA, Salvatore Frasca J, Rood D et al (2005) Correlates of immune protection in chickens vaccinated with Mycoplasma gallisepticum strain GT5 following challenge with pathogenic M. gallisepticum strain rlow. Infect Immun 73(9):5410–5419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Liu M, Luo Y, Tao R et al (2009) Sensitive and rapid detection of genetic modified soybean (roundup ready) by loop-mediated isothermal amplification. Biosci Biotechnol Biochem 73(11):2365–2369

    Article  CAS  PubMed  Google Scholar 

  4. Kusum S, Reema B, Aman S et al (2014) Loop-mediated isothermal amplification for rapid diagnosis of tubercular uveitis. Jama Ophthalmol 132(132):777–778

    Google Scholar 

  5. Scheel CM, Zhou Y, Theodoro RC et al (2014) Development of a loop-mediated isothermal amplification method for detection of histoplasma capsulatum DNA in clinical samples. J Clin Microbiol 52(2):483–488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hassan M, Vittal R, Raj JM et al (2022) Loop-mediated isothermal amplification (LAMP): a sensitive molecular tool for detection of Staphylococcus aureus in meat and dairy product. Braz J Microbiol 53(1):341–347

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hara-Kudo Y, Konishi N, Ohtsuka K et al (2008) Detection of verotoxigenic Escherichia coli O157 and O26 in food by plating methods and LAMP method: a collaborative study. Int J Food Microbiol 122(1–2):156–161

    Article  CAS  PubMed  Google Scholar 

  8. Pang B, Yao S, Xu K et al (2019) A novel visual-mixed-dye for LAMP and its application in the detection of foodborne pathogens. Anal Biochem 574:1–6

    Article  CAS  PubMed  Google Scholar 

  9. Zhu L, Xu Y, Cheng N et al (2017) A facile cascade signal amplification strategy using DNAzyme loop-mediated isothermal amplification for the ultrasensitive colorimetric detection of Salmonella. Sens Actuators, B Chem 242:880–888

    Article  CAS  Google Scholar 

  10. Notomi T, Okayama H, Masubuchi H et al (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):E63

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Azizi M, Zaferani M, Cheong SH et al (2019) Pathogenic bacteria detection using RNA-based loop-mediated isothermal-amplification-assisted nucleic acid amplification via droplet microfluidics. Acs Sensors 4(4):841–848

    Article  CAS  PubMed  Google Scholar 

  12. Kokkinos PA, Ziros PG, Bellou M et al (2014) Loop-mediated isothermal amplification (LAMP) for the detection of salmonella in food. Food Anal Methods 7:512–526

    Article  Google Scholar 

  13. Mori Y, Notomi T (2009) Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 15(2):62–69

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Dickinson M, Hodgetts J (2013) In-field diagnostics using loop-mediated isothermal amplification. Methods Mol Biol 938:291–300. https://doi.org/10.1007/978-1-62703-089-2_25

    Article  CAS  Google Scholar 

  15. Patel JC, Lucchi NW, Srivastava P et al (2014) Field evaluation of a real-time fluorescence loop-mediated isothermal amplification assay, RealAmp, for the diagnosis of malaria in Thailand and India. J Infect Dis 8:1180–1187

    Article  Google Scholar 

  16. Njiru ZK, Büscher P (2012) Loop-mediated isothermal amplification technology: towards point of care diagnostics. PLoS Negl Trop Dis 6(6):e1572

    Article  PubMed  PubMed Central  Google Scholar 

  17. Fang X, Liu Y, Kong J et al (2010) Loop-mediated isothermal amplification integrated on microfluidic chips for point-of-care quantitative detection of pathogens. Anal Chem 82(7):3002

    Article  CAS  PubMed  Google Scholar 

  18. Safavieh M, Kanakasabapathy MK, Tarlan F et al (2016) Emerging loop-mediated isothermal amplification-based microchip and microdevice technologies for nucleic acid detection. ACS Biomater Sci Eng 2(2):278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mori Y, Nagamine K, Tomita N et al (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289(1):150–154

    Article  CAS  PubMed  Google Scholar 

  20. Gray CM, Katamba A, Narang P et al (2016) Feasibility and operational performance of tuberculosis detection by loop-mediated isothermal amplification platform in decentralized settings: results from a multicenter study. J Clin Microbiol 54(8):1984–1991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16(3):223–229

    Article  CAS  PubMed  Google Scholar 

  22. Parida M, Horioke K, Ishida H et al (2005) Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay. J Clin Microbiol 43(6):2895–2903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Goto M, Honda E, Ogura A et al (2009) colorimetric detection of loop- mediated isothermal ampli cation reaction by using hydroxy naphthol blue. Biotechniques 46(3):167–172

    Article  CAS  PubMed  Google Scholar 

  24. Mahony J, Chong S et al (2013) Development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in 30 minutes. J Clin Microbiol 51(8):2696–2701

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Imai M, Ai N, Minekawa H et al (2007) Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5 hemagglutinin gene-specific loop-mediated isothermal amplification method. J Virol Methods 141(2):173–180

    Article  CAS  PubMed  Google Scholar 

  26. Zhang X, Xu G, Tang H et al (2017) Development of loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Alternaria alternata. J AOAC Int 100(1):99–103

    Article  CAS  PubMed  Google Scholar 

  27. Wen H, Hao Z, Xu J et al (2017) Loop-mediated isothermal amplification method for the rapid detection of Ralstonia solanacearum phylotype I mulberry strains in China. Front Plant 8:76

    Google Scholar 

  28. Lucchi NW, Gaye M, Diallo MA et al (2016) Evaluation of the illumigene malaria LAMP: a robust molecular diagnostic tool for malaria parasites. Sci Rep 6:36808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hirayama H, Kageyama S, Takahashi Y et al (2006) Rapid sexing of water buffalo (Bubalus bubalis) embryos using loop-mediated isothermal amplification. Theriogenology 66(5):1249–1256

    Article  CAS  PubMed  Google Scholar 

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Funding

The work described in this paper was fully supported by a grant from the High-level Science and Technology Innovation and Entrepreneurship Talent Project of Shijiazhuang (No. 05202001).

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Authors and Affiliations

  1. College of Life Science, Hebei Agricultural University, Baoding, 071000, Hebei, China

    Yu Liu & Man Wang

  2. Institute of Food Safety and Nutrition, Jinan University, Guangzhou, 510632, Guangdong, China

    Yilin Zhang, Lei Ye & Lei Shi

  3. Hebei Sanshi Biotechnology Co., Ltd., No. 136, Huanghe Avenue, Shijiazhuang, 050035, Hebei, China

    Guoqiang Shi, Zhenguo Dong & Lei Shi

Authors
  1. Yu Liu
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  2. Yilin Zhang
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  3. Man Wang
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  4. Guoqiang Shi
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  5. Zhenguo Dong
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  6. Lei Ye
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  7. Lei Shi
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Contributions

YL developed the TaqMan-LAMP method and prepared the original draft; YZ and MW treated the samples; ZD, LY and LS edited the manuscript; and GS acquired funding. All the authors have read and agreed to the published version of the manuscript.

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Correspondence to Lei Ye or Lei Shi.

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Liu, Y., Zhang, Y., Wang, M. et al. Modified loop-mediated isothermal amplification method combined with a TaqMan probe for the detection of Mycoplasma gallisepticum. Eur Food Res Technol 249, 1469–1477 (2023). https://doi.org/10.1007/s00217-023-04226-9

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  • DOI: https://doi.org/10.1007/s00217-023-04226-9

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