Isothermal loop-mediated amplification (lamp) for diagnosis of contagious bovine pleuro-pneumonia

BMC Veterinary Research

Hermann Unger¹  Joachim Frey⁴  Abel Wade³  Edy M Vilei⁵  Georg Mair² 

[1] Animal Production and Health Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Wagramer Strasse 5, P.O. Box 100, Vienna A-1400, Austria;Tropical Vet. Med. Laboratory, University of Veterinary Medicine, Vienna, Austria;LANAVET, Garoua, Cameroun;Institute of Veterinary Bacteriology, Vetsuisse, University of Bern, Bern, Switzerland;Current address: Suisselab AG, Schützenstrasse 10, Zollikofen, BE, CH-3052, Switzerland

关键词: Field test;    Molecular diagnostic;    Loop-mediated amplification LAMP;    Isothermal;    CBPP Mycoplasma mycoides;   

Others  :  1119530
DOI  :  10.1186/1746-6148-9-108

 received in 2013-01-21, accepted in 2013-05-14,  发布年份 2013

【 摘 要 】

Background

Contagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clinical diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labour-intensive bacteriological methods for the detection of M. mycoides subsp. mycoides have been replaced by molecular genetic techniques in the last two decades, these latter approaches require well-equipped laboratories and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential.

Results

We present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clinical cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/pleural fluids and serum/plasma within an hour using a simple dilution protocol. A photometric detection of LAMP products allows the real-time visualisation of the amplification curve and the application of a melting curve/re-association analysis presents a means of quality assurance based on the predetermined strand-inherent temperature profile supporting the diagnosis.

Conclusion

The CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clinical or post mortem samples. The stringent innate quality control allows a conclusive on-site diagnosis of CBPP such as during farm or slaughter house inspections.

【 授权许可】

   
2013 Mair et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Manso-Silván L, Vilei EM, Sachse K, Djordjevic SP, Thiaucourt F, Frey J: Mycoplasma leachii sp. nov. as a new species designation for Mycoplasma sp. bovine group 7 of Leach, and reclassification of Mycoplasma mycoides subsp. mycoides LC as a serovar of Mycoplasma mycoides subsp. capri. Int J Syst Evol Microbiol 2009, 59:1353-1358.
• [2]Roeder PL: Rinderpest: The end of cattle plague. Prev Vet Med 2011, 102:98-106.
• [3]Mbulu RS, Tjipura-Zaire G, Lelli R, Frey J, Pilo P, Vilei EM, Mettler F, Nicholas RA, Huebschle OJ: Contagious bovine pleuropneumonia (CBPP) caused by vaccine strain T1/44 of Mycoplasma mycoides subsp. mycoides SC. Vet Microbiol 2004, 98:229-234.
• [4]Thiaucourt F, Aboubakar Y, Wesonga H, Manso-Silván L, Blanchard A: Contagious bovine pleuropneumonia vaccines and control strategies: recent data. Dev Biol (Basel) 2004, 119:99-111.
• [5]Wesonga H, Manso-Silván L, Thiaucourt F: CBPP vaccine strain T1/44: possible reversion to virulence. Proceedings of the 3rd Meeting of the FAO/OIE/OAU-IBAR/IAEA Consultative Group in CBPP in Africa. Rome. Rome: FAO Publications Rome; 2003.
• [6]Amanfu W, Sediadie S, Masupu KV, Raborokgwe MV, Benkirane A, Geiger R, Thiaucourt F: Comparison between c-ELISA and CFT in detecting antibodies to Mycoplasma mycoides mycoides biotype SC in cattle affected by CBPP in Botswana. Ann N Y Acad Sci 2000, 916:364-369.
• [7]Bruderer U, Regalla J, Abdo E-M, Huebschle OJB, Frey J: Serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP) with an indirect ELISA based on the specific lipoprotein LppQ of Mycoplasma mycoides subsp. mycoides SC. Vet Microbiol 2002, 84:195-205.
• [8]Le Goff C, Thiaucourt F: A competitive ELISA for the specific diagnosis of contagious bovine pleuropneumonia (CBPP). Vet Microbiol 1998, 60:179-191.
• [9]Muuka G, Hang’ombe BM, Nalubamba KS, Kabilika S, Mwambazi L, Muma JB: Comparison of complement fixation test, competitive ELISA and LppQ ELISA with post-mortem findings in the diagnosis of contagious bovine pleuropneumonia (CBPP). Trop Anim Health Prod 2011, 43:1057-1062.
• [10]Schubert E, Sachse K, Jores J, Heller M: Serological testing of cattle experimentally infected with Mycoplasma mycoides subsp. mycoides small colony using four different tests reveals a variety of seroconversion patterns. BMC Vet Res 2011, 7:72. BioMed Central Full Text
• [11]Tardy F, Gaurivaud P, Manso-Silván L, Thiaucourt F, Pellet MP, Mercier P, Le Grand D, Poumarat F: Extended surveillance for CBPP in a free country: Challenges and solutions regarding the potential caprine reservoir. Prev Vet Med 2011, 101:89-95.
• [12]Bashiruddin JB, Frey J, Königsson MH, Johansson KE, Hotzel H, Diller R, De Santis P, Botelho A, Ayling RD, Nicholas RA, Thiaucourt F, Sachse K: Evaluation of PCR systems for the identification and differentiation of Mycoplasma agalactiae and Mycoplasma bovis: a collaborative trial. Vet J 2005, 169:268-275.
• [13]Fitzmaurice J, Sewell M, Manso-Silván L, Thiaucourt F, McDonald WL, O’Keefe JS: Real-time polymerase chain reaction assays for the detection of members of the Mycoplasma mycoides cluster. N Z Vet J 2008, 56:40-47.
• [14]Lorenzon S, Manso-Silván L, Thiaucourt F: Specific real-time PCR assays for the detection and quantification of Mycoplasma mycoides subsp. mycoides SC and Mycoplasma capricolum subsp. capripneumoniae. Mol. Cell. Probes 2008, 22:324-328.
• [15]Miserez R, Pilloud T, Cheng X, Nicolet J, Griot C, Frey J: Development of a sensitive nested PCR method for the specific detection of Mycoplasma mycoides subsp. mycoides SC. Mol Cell Probes 1997, 11:103-111.
• [16]Schnee C, Heller M, Heller M, Jores J, Tomaso H, Neubauer H: Assessment of a novel multiplex real-time PCR assay for the detection of the CBPP agent Mycoplasma mycoides subsp. mycoides SC through experimental infection in cattle. BMC Vet Res 2011, 7:47. BioMed Central Full Text
• [17]Vilei EM, Frey J: Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ¯ mutant vaccine strain used for DIVA-strategies. J Microbiol Methods 2010, 81:211-218.
• [18]Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T: Loop-mediated isothermal amplification of DNA. Nucl. Acids Res. 2000, 28:E63.
• [19]Mabey D, Peeling RW, Ustianowski A, Perkins MD: Diagnostics for the developing world. Nat Rev Microbiol 2004, 2(3):231.
• [20]Pilo P, Vilei EM, Peterhans E, Bonvin-Klotz L, Stoffel MH, Dobbelaere D, Frey J: A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony. J Bacteriol 2005, 187:6824-6831.
• [21]Westberg J, Persson A, Holmberg A, Goesmann A, Lundeberg J, Johansson KE, Pettersson B, Uhlen M: The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Res 2004, 14:221-227.
• [22]Breslauer KJ, Frank R, Marky LA BH: Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci USA 1986, 83(11):3746-3750.
• [23]Steger G: Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction. Nucleic Acids Res 1994, 22(14):2760-2768.
• [24]Gorton TS, Barnett MM, Gull T, French RA, Lu Z, Kutish GF, Adams LG, Geary SJ: Development of real-time diagnostic assays specific for Mycoplasma mycoides subspecies mycoides Small Colony. Vet Microbiol 2005, 111:51-58.
• [25]Schütz E, Von Ahsen N: Spreadsheet software for thermodynamic melting point prediction of oligonucleotide hybridization with and without mismatches. Biotechniques 1999, 27(6):1218-1224.
• [26]Kibbe WA: OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res 2007, 35(Web Server Issue):43-46.
• [27]Savitzky A, Golay MJE: Smoothing and differentiation of data by simplified least squares procedures. analytical chemistry 1964, S. 1627-S. 1639. 36, Nr. 8, 1. Juni