Rapid Diagnosis of Bovine Brucellosis by Loop Mediated Isothermal Amplification (LAMP) assay

Öz

Brucellosis is a zoonotic infection that cause abortions and infertility in livestock by effecting of especially genital organs like uterus, testes, mammary glands. In this study, it was aimed to use loop mediated isothermal amplification (LAMP), which is sensitive and specific molecular technique that based on amplification of BCSP31 gene, which is unique to organisms in the genus Brucella. In this study, a total of 57 materials including 20 milk samples and 37 aborted fetuses were tested by LAMP and classical bacterial culture for diagnosis of brucellosis. Thirteen (22.8%) out of 57 samples were found positive by LAMP while 10 samples (17.5%) yielded bacterial growth. As a conclusion, LAMP could be used satisfactorily for the diagnosis of brucellosis and the sensitivity can be increased by using two techniques together. We concluded that the bacterial culture is still gold standard for diagnosis of brucellosis although the sensitivity and specificity of the LAMP is high. 

Anahtar Kelimeler

• Brucellosis,cattle,LAMP (Loop Mediated Isothermal Amplification),molecular diagnosis

Loop Mediated Isothermal Amplification (LAMP) Yöntemi ile Sığır Brusellozunun Hızlı Teşhisi

Öz

Bruselloz, sığır, koyun, keçi, domuz ve koç gibi hayvanlarda, özellikle, testis, meme, uterus gibi genital organlara yerleşerek yavru atmalara ve infertiliteye neden olan zoonoz bir hastalıktır. Bu çalışmada bruselloz hastalığının çabuk tanısında BCSP31 genini temel alan duyarlı ve özgül özel bir moleküler teknik olan ilmek aracılı bir gen çoğaltma tekniğinin (loop mediated isothermal amplification), kullanılması amaçlandı. Bruselloz şüpheli hayvanlardan alınan 20 adet süt ve 37 adet aborte fötüs olmak üzere toplam 57 adet materyal LAMP ve kültür yöntemiyle incelendi. İncelenen materyallerin 13 adedi (%22,8) LAMP ile pozitif bulunurken, kültür yöntemi ile örneklerin 10’undan (%17,5) etken izole ve identifiye edildi. Sonuç olarak, LAMP tekniğinin brusellozun teşhisinde güven ile kullanılabileceği, LAMP testiyle beraber kültür yönteminin uygulanmasının teşhis sensitivitesinin artırılmasında yararlı olduğu saptandı. LAMP testinin spesifite ve sensitivitesinin yüksek olmasına rağmen hala kültür yönteminin altın standart olarak geçerliliğini sürdürdüğü düşünülmektedir.

Anahtar Kelimeler

• Bruselloz,LAMP (Loop Mediated İsothermal Amplification),moleküler teşhis,sığır

Kaynakça

1. 1. Corbel MJ. (1997). Brucellosis: an overview. Emerg Infect Dis. 3(2): 213-221.
2. 2. Blasco JM. (1990). Brucella Ovis. In: Animal Brucello-sis. Nielsen K, Duncan JR, (eds). pp. 351–378. CRC Press, Boca Raton, Florida, USA.
3. 3. Alton GG, Jones LM, Angus RD, Verger JM. (1988). Techniques for The Brucellosis Laboratory. I.N.R.A, Paris, France.
4. 4. Notomi T, Okayama H, Masubuchi H, et al. (2000). Loop-Mediated Isothermal Amplification of DNA. Nucleic Acids Res. 28(12): e63.
5. 5. Song L, Li J, Hou S, Li X, Chen S. (2012). Establishment of Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection of Brucella spp. and Application to Milk and Blood Samples. J Microbiol Methods. 90: 292-297.
6. 6. Lin GZ, Zheng FY, Zhou JZ, et al (2011). Loop-Mediated Isothermal Amplification Assay Targeting the omp25 Gene for Rapid Detection of Brucella spp. Mol Cell Probes. 25: 126-129.
7. 7. Misawa Y, Yoshida A, Saito R, et al. (2007). Application of Loop-mediated Isothermal Amplification Technique to Rapid and Direct Detection of Methicillin resistant Staphylococcus aureus (MRSA) in Blood Cultures. J Infect Chemother. 13(3): 134-140.
8. 8. Tao ZY, Zhou HY, Xia H, Xu S, Zhu HW. (2011). Adaptation of a Visualized Loop-Mediated Isothermal Amplification Technique for Field Detection of Plasmodium vivax Infection. Parasites Vect. 4(1): 115.

9. 9. Wang L, Li Y, Chu J, Xu Z, Zhong Q. (2010). Devel-opment and Application of a Simple Loop-mediated Isothermal Amplification Method on Rapid Detection of Listeria monocytogenes strains. Molecular Biol Reports. 39(1): 445-449.
10. 10. Yamazaki W, Taguchi M, Ishibashi M, et al. (2008). Development and Evaluation of a Loop-mediated Isothermal Amplification Assay for Rapid and Simple Detection of Campylobacter jejuni and Campylobacter coli. J Med Microbiol. 57(4): 444-451.
11. 11. OIE (2009). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Sixth Edition, Caprine and Ovine Brucellosis. Pp.1022-1030.
12. 12. Ohtsuki R, Kawamoto K, Kato Y, Shah MM, Ezaki T, Makino SI. (2008). Rapid Detection of Brucella spp. by the Loop‐mediated Isothermal Amplification Method. J Appl Microbiol. 104(6): 1815-1823.
13. 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: 62–69.
14. 14. Saharan P, Dhingolia S, Khatri P, Duhan JS, Gahlawat SK. (2014). Loop-Mediated Isothermal Amplification (LAMP) Based Detection of Bacteria: A review. Afr J Biotechnol. 13(9): 1920-1928.
15. 15. Pérez-Sancho M, García-Seco T, Arrogante L, et al. (2013). Development and Evaluation of an IS711-based Loop-mediated Isothermal Amplification Method (LAMP) for Detection of Brucella spp. on Clinical Samples. Res Vet Sci. 95(2), 489-494.
16. 16. Karthik K, Rathore R, Thomas P, et al. (2014). Loop-Mediated Isothermal Amplification (LAMP) Test for Specific and Rapid Detection of Brucella abortus in Cattle. Vet Q. 34 (4): 174-179.
17. 17. Batinga MC, de Lima JTR, Gregori F, et al. (2018). Comparative Application of Is711-Based Polymerase Chain Reaction (PCR) and Loop-Mediated Isothermal Amplification (LAMP) for Canine Brucellosis Diagnosis. Molecular and Cellular Probes Mol Cell Probes. 39:1-6.
18. 18. Kang SI, Her M, Kim YJ, et al. (2015). Rapid and Specific Identification of Brucella Abortus Using the Loop-Mediated Isothermal Amplification (LAMP) Assay. Comp Immunol Microbiol Infect Dis. 40: 1-6.