Development of LAMP-Lateral Flow Immunoassay for diagnosis of melioidosis


  • Priyapa Najomtien Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
  • Chotima Potisap Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
  • Supaksorn Chattagul Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
  • Rasana W. Sermswan Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
  • Surasak Wongratanacheewin Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.


Melioidosis, Laboratory diagnosis, LAMP


Background and Objective: Melioidosis is a fatal disease caused by a gram-negative bacterium, Burkholderia pseudomallei. Patients with septicemic melioidosis die within 48 hours, the rapid diagnosis using blood samples is essential. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick (LFD) assays for the detection of B. pseudomallei.

Methods: The wcbG gene, a putative capsular polysaccharide biosynthesis protein gene of B. pseudomallei was selected for LAMP primers design. Four primers and 1 probe were considered using Primer Explorer software. The conditions for LAMP-LFD were optimized. The sensitivity of LAMP-LFD was investigated with 50 ng to 500 fg of B. pseudomallei genomic DNA while the specificity was evaluated by using 50 ng of 12 bacterial strains. The LAMP-LFD method was evaluated by the spiked 106 CFU to 1 CFU of B. pseudomallei into the normal EDTA blood samples.

Results: The LAMP-LFD was successfully developed using 1M betaine, 60°C for 60 min for LAMP cycles and conditions for hybridization with FITC-probe were at 60°C for 5 min. It gave sensitivity of 5 pg/ml and 100% specificity when tested with B. thailandensis, Staphylococcus aureus, S. epidermidis, Escherichia coli, Salmonella group B, Pseudomonas aeruginosa and Klebsiella pneumonia. The detection limit was genomic DNA of 102 CFU per 200 μl of EDTA blood.

Conclusion: Our LAMP-LFD assay provided a highly sensitive and specific method to detect B. pseudomallei in the blood based on wcbG gene. It will be another approach for rapid diagnosis of melioidosis.


Wiersinga WJ, Virk HS, Torres AG, Currie BJ, Peacock SJ, Dance DA, et al. Melioidosis. Nat Rev Dis Primers 2018;4(1):17107. doi:10.1038/nrdp.2017.107

Limmathurotsakul D, Wongratanacheewin S, Teerawattanasook N, Wongsuvan G, Chaisuksant S, Chetchotisakd P. et al. Increasing incidence of human melioidosis in northeast Thailand. Am J Trop Med Hyg 2010;82(6):1113-7. doi:10.4269/ ajtmh.2010.10-0038

Houghton RL, Reed DE, Hubbard MA, Dillon MJ, Chen H, Currie BJ, et al. Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis. PLoS Negl Trop Dis 2014;8(3):e2727. doi:10.1371/journal.pntd.0002727

Wongsuvan G, Hantrakun V, Teparrukkul P, Imwong M, West TE, Wuthiekanun V, et al. Sensitivity and specificity of a lateral flow immunoassay (LFI) in serum samples for diagnosis of melioidosis. Trans R Soc Trop Med Hyg 2018;112(12):568-70. doi:10. 1093/trstmh/try099

Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28(12):e63. doi:10.1093/nar/28.12.e63

Chantratita N, Meumann E, Thanwisai A, Limmathurotsakul D, Wuthiekanun V, Wannapasni S, et al. Loop-mediated isothermal amplification method targeting the TTS1 gene cluster for detection of Burkholderia pseudomallei and diagnosis of melioidosis. J Clin Microbiol 2008;46(2): 568–73. doi:10.1128/JCM.01817-07

Sermswan RW, Royros P, Khakhum N, Wongratana¬cheewin S, Tuanyok A. Direct detection of Burkholderia pseudomallei and biological factors in soil. Trans R Soc Trop Med Hyg 2015;109(7): 462–8. doi:10.1093/trstmh/trv040

Anderson DG, McKay LL. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol 1983;46(3): 549-52. doi:10.1128/aem.46.3.549-552.1983

Rattanathongkom A, Sermswan RW, Wongratana¬cheewin S. Detection of Burkholderia pseudom¬allei in blood samples using polymerase chain reaction. Mol Cell Probes 1997;11(1):25–31. doi: 10.1006/mcpr.1996.0072

Jin JL, Ning YX. Septicemic melioidosis: a case report and literature review. J Thorac Dis 2014;6(2): E1-4. doi:10.3978/j.issn.2072-1439.2014.01.20

Warapitiya DS, Subasinghe S, Silva RF De, Piyarisi DL, Jayatilleke K. Severe Sepsis with Multiorgan Failure due to Melioidosis: A Lesson to Learn. Case Rep Med 2021;8(1):1-5. doi:10.1155/2021/5563214

Deris ZZ, Hasan H, Suraiya MNS. Clinical characteristics and outcomes of bacteraemic melioidosis in a teaching hospital in a northeastern state of Malaysia: A five-year review. Vol. 4, J Infect Dev Ctries 2010;4(7):430-5. doi:10.3855/jidc.491

Goodyear A, Strange L, Rholl DA, Silisouk J, Dance DAB, Schweizer HP, et al. An improved selective culture medium enhances the isolation of Burkholderia pseudomallei from contaminated specimens. Am J Trop Med Hyg 2013;89(5):973–82. doi:10.4269/ajtmh.13-0119

Nhem S, Letchford J, Meas C, Thann S, McLaughlin JC, Baron EJ, et al. Detection of Burkholderia pseudomallei in Sputum using Selective Enrichment Broth and Ashdown’s Medium at Kampong Cham Provincial Hospital, Cambodia. F1000Res 2014;3(302):1-5. doi:10.12688/ f1000research. 5935.1

Opota O, Jaton K, Greub G. Microbial diagnosis of bloodstream infection: Towards molecular diagnosis directly from blood. Clin Microbiol Infect 2015;21(4):323–31. doi:10.1016/j.cmi.2015 .02.005

Palasatien S, Lertsirivorakul R, Royros P, Wongratanacheewin S, Sermswan RW. Soil physicochemical properties related to the presence of Burkholderia pseudomallei. Trans R Soc Trop Med Hyg 2008;102(Suppl 1):S5-9. doi:10.1016/S0035-9203(08)70003-8

Wang Y, Li H, Wang Y, Zhang L, Xu J, Ye C. Loop-mediated isothermal amplification label-based gold nanoparticles lateral flow biosensor for detection of Enterococcus faecalis and Staphylococcus aureus. Front Microbiol 2017;10(8):1-14. doi:10.3389/fmicb.2017.00192

Zhang X, Lowe SB, Gooding JJ. Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP). Biosens Bioelectron 2014;15(61):491–9. doi:10.1016/j.bios.2014.05.039

Liu L, Xu Y, Zhong W, Li L, Li W, Xiao Q. Comparison of three terminal detection methods based on loop mediated isothermal amplification (Lamp) assay for spring viremia of carp virus (svcv). Turk J Fish Aquat Sci 2019;19(9):805–16. doi:10.4194/ 1303-2712-v19_9_09

Zhang J, Cao J, Zhu M, Xu M, Shi F. Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Mycoplasma ovipneumoniae. World J Microbiol Biotechnol 2019; 35(2). doi:10.1007/s11274-019-2601-5

Kumvongpin R, Jearanaikoon P, Wilailuckana C, Sae-Ung N, Prasongdee P, Daduang S, et al. Detection assay for HPV16 and HPV18 by loop-mediated isothermal amplification with lateral flow dipstick tests. Mol Med Rep 2017;15(5): 3203–9. doi:10.3892/mmr.2017.6370

Gong P, Zhang T, Chen F, Wang L, Jin S, Bai X. Advances in loop-mediated isothermal amplification: Integrated with several point-of-care diagnostic methods. Analytical Methods 2014;19(6): 7585–9. doi:10.1039/C4AY00330F

Mori Y, Hirano T, Notomi T. Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC Biotechnol 2006;6(3):1-10. doi:10.1186/1472-6750-6-3

Kaewphinit T, Arunrut N, Kiatpathomchai W, Santiwatanakul S, Jaratsing P, Chansiri K. Detection of Mycobacterium tuberculosis by using loop-mediated isothermal amplification combined with a lateral flow dipstick in clinical samples. Biomed Res Int 2013;2013(8671):1-7. doi: 10.1155/2013/ 926230

Khunthong S, Jaroenram W, Arunrut N, Suebsing R, Mungsantisuk I, Kiatpathomchai W. Rapid and sensitive detection of shrimp yellow head virus by loop-mediated isothermal amplification combined with a lateral flow dipstick. J Virological Methods 2013;188(1-2):51–6. doi: 10.1016/j.jviromet. 2012.11.041




How to Cite

Najomtien P, Potisap C, Chattagul S, W. Sermswan R, Wongratanacheewin S. Development of LAMP-Lateral Flow Immunoassay for diagnosis of melioidosis. SRIMEDJ [Internet]. 2024 Apr. 24 [cited 2024 Jun. 19];39(2):156-6. Available from:



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