Development of a rapid, one-step-visual method to detect Salmonella based on IC-LAMP method

Document Type : Full paper (Original article)

Authors

1 MSc Student in Biochemical Engineering, Molecular Medicine Research Center of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China

2 Molecular Medicine Research Center of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China

Abstract

Background: Salmonella can cause serious human gastroenteritis and is frequently isolated from various food samples. The cell culturing, immunoassay, and polymerase chain reactions (PCR) are the current methods to detect such pathogenic agents. However, these methods are time-consuming and labor-intensive, and thus unavailable for rapid-monitoring of Salmonella. Aims: This study aimed to develop an immunocapture-loop-mediated isothermal amplification (IC-LAMP) for rapid and sensitive detection of Salmonella. Methods: Salmonella was used as antigen to produce monoclonal antibody (mAb) and mAbs were prepared via subcloning three times. The mAb 1B12 with high affinity was coated on the surface of the immuno-magnetic beads (IMBs) to capture Salmonella. The enriched products (IMBs-Salmonella) were used for LAMP using the special primers targeted the conserved invA gene of Salmonella. Results: The IC-LAMP was developed based on mAb 1B12 and LAMP. Targeting the conserved invA gene of Salmonella, the detection time was shortened to 50 min from three days. If the reaction contains Salmonella, the green fluorescence and the trapezoidal strip can be clearly observed. Importantly, the method combines the specificity of antibody and LAMP with a detection limit of 5 CFU/ml in artificially contaminated water and milk. The specificity of this method was demonstrated by testing other similar bacteria. The results indicate that the IC-LAMP reacts only with Salmonella and does not cross-react with other similar bacteria. Conclusion: The IC-LAMP assay developed here is a rapid, sensitive, one-step-visual method to screen for the presence of Salmonella in food samples. This method is faster than traditional PCR, LAMP, and other methods, and can be used as a primary screening method for the detection.

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Main Subjects

References

Alves, J; Niguma, NH and de Oliveira, TCRM (2015). Detection of Salmonella spp. in eight complex food matrices using polymerase chain reaction assay. J. Food Safety. 35: 453-457.
Chin, WH; Sun, Y; Hogberg, J; Quyen, TL; Engelsmann, P; Wolff, A and Bang, DD (2017). Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples. Mol. Cell. Probes., 32: 24-32.
Datta, S; Dandapat, P and Jas, R (2018). Diagnosis of mixed gastrointestinal nematode infection in goat by an indirect-ELISA. Iran. J. Vet. Res., 19: 189-193.
Deb, R; Sengar, GS; Singh, U; Kumar, S; Raja, TV; Alex, R; Alyethodi, RR and Prakash, B (2017). LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples. Iran. J. Vet. Res., 18: 134-137.
Du, W; Wang, Y; Huang, L; Wei, Y; Chen, D; Sun, J; Wu, H; Feng, L and Liu, C (2016). Characterization of monoclonal antibodies that recognize the amino- and carboxy-terminal epitopes of the pseudorabies virus UL42 protein. Appl. Microbiol. Biotechnol., 100: 181-192.
Elzein, M; Alum, A and Abbaszadegan, M (2013). Biochemical signature assay for use in a biosensor platform to detect bacteria in drinking water biofilms. Environ. Lett., 48: 925-932.
Encheng, S; Jing, Z; Tao, Y; Qingyuan, X; Yongli, Q; Wenshi, W; Peng, W; Liang, S; Jing, S and Donglai, W (2013). Analysis of murine B-cell epitopes on Eastern equine encephalitis virus glycoprotein E2. Appl. Microbiol. Biotechnol., 97: 6359-6372.
Handley, JA; Shi, Z; Si, HP; Dawoud, TM; Kwon, YM and Ricke, SC (2015). Salmonella and the potential role for methods to develop microbial process indicators on chicken carcasses. Food safety., PP: 81-104.
Hapuarachchi, CT; Jeffery, KJM and Bowler, ICJW (2019). Stool PCR may not be a substitute for enrichment culture for the detection of Salmonella. J. Med. Microbiol., 68: 395-397.
Hemmatzadeh, F and Kazemimanesh, M (2017). Detection of specific antigens of Newcastle disease virus using an absorbed western blotting method. Iran. J. Vet. Res., 18: 92-96.
Kanitkar, YH; Stedtfeld, RD; Hatzinger, PB; Hashsham, SA and Cupples, AM (2017). Development and application of a rapid, user-friendly, and inexpensive method to detect Dehalococcoides sp. reductive dehalogenase genes from ground water. Appl. Microbiol. Biotechnol., 101: 4827-4835.
Karp, BE; Campbell, D; Chen, JC; Folster, JP and Friedman, CR (2018). Plasmid-mediated quinolone resistance in human non-typhoidal Salmonella infections: an emerging public health problem in the United States. Zoonoses. Public. Hlth., 65: 838-849.
Khan, RSA; Ali, W; Kiran, S; Shah, MSD; Tahir, ZA and Habib, M (2018). Rapid detection of infectious bursal disease by loop-mediated isothermal amplification for field analysis. Iran. J. Vet. Res., 19: 101-107.
Law, JW; Ab Mutalib, NS; Chan, KG and Lee, LHM (2014). Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations. Front. Microbiol., 5: 770.
Liu, S; Geng, Y; Liu, L; Sun, X; Shao, J; Han, B; Wang, J and Tan, K (2018). Development of an isothermal amplification-based assay for the rapid detection of Cronobacter spp. J. Dairy Sci., 101: 4914-4922.
Majowicz, SE; Musto, J; Scallan, E; Angulo, FJ; Kirk, M; O’Brien, SJ; Jones, TF; Fazil, A and Hoekstra, RM (2010). The global burden of nontyphoidal Salmonella gastroenteritis. Clin. Infect. Dis., 50: 882-889.
Mori, Y; Nagamine, K; Tomita, N and Notomi, T (2001). Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun., 289: 150-154.
Mori, Y and 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.
Notomi, T; Okayama, H; Masubuchi, H; Yonekawa, T; Watanabe, K; Amino, N and Hase, T (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids. Res., 28: E63.
Royer, S and Pare, D (2003). Conservation of total synaptic weight through balanced synaptic depression and potentiation. Nature. 422: 518-522.
Salzman, NH; Ghosh, D; Huttner, KM; Paterson, Y and Bevins, CL (2003). Protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin. Nature. 422: 522-526.
Shehata, DM and Lu, X (2016). Development of a loop mediated isothermal amplification (LAMP) - surface enhanced raman spectroscopy (SERS) assay for the detection of Salmonella enterica serotype enteritidis. Theranostics. 6: 522.
Singh, G; Sithebe, A; Enitan, AM; Kumari, S; Bux, F and Stenstrom, TA (2017). Comparison of droplet digital PCR and quantitative PCR for the detection of Salmonella and its application for river sediments. J. Water Health. 15: 505-508.
Song, J; Mauk, MG; Hackett, BA; Cherry, S; Bau, HH and Liu, C (2016). Instrument-free point-of-care molecular detection of Zika virus. Anal. Chem., 88: 7289-7294.
Soria, MC; Soria, MA; Bueno, DJ and Terzolo, HR (2013). Comparison of 3 culture methods and PCR assays for Salmonella gallinarum and Salmonella pullorum detection in poultry feed. Poult. Sci., 92: 1505-1515.
Sun, J; Huang, L; Wei, Y; Wang, Y; Chen, D; Du, W; Wu, H; Feng, L and Liu, C (2015). Identification of three PPV1 VP2 protein-specific B cell linear epitopes using monoclonal antibodies against baculovirus-expressed recombinant VP2 protein. Appl. Microbiol. Biotechnol., 99: 9025-9036.
Xia, T; Jia, C; Ying, W and Wang, X (2016). Gene cloning, expression and polyclonal antibody preparation of Rab3A for protein interaction analysis. Springer plus., 5: 1705.
Zhang, L; Du, X; Chen, C; Chen, Z; Gong, N; Song, Y; Song, Y; Han, Q; Xia, X;Luo, H and Zhang, J (2019). Instrument-Free and Visual Detection of Salmonella Based
on Magnetic Nanoparticles and an Antibody Probe Immunosensor. Int. J. Mol. Sci., 20(18).
Zhang, S; Kingsley, RA; Santos, RL; Andrews-Polymenis, H; Raffatellu, M; Figueiredo, J; Nunes, J; Tsolis, RM; Adams, LG and Baumler, AJ (2003). Molecular pathogenesis of Salmonella enterica serotype Typhimurium-induced diarrhea. Infect. Immun., 71: 1-12.
Zhang, L; Shi, Y; Chen, C; Han, Q; Chen, Q; Xia, X; Song, Y and Zhang, J (2019). Rapid, visual detection of Klebsiella pneumoniae using magnetic nanoparticles and an horseradish peroxidase-probe based immunosensor. J. Biomed. Nanotechnol., 15: 1061-1071.
Zhang, L; Shi, Y; Chen, C; Han, Q; Zhang, M; Yi, H; Song, Y; Xia, X and Zhang, J (2019). Visual and rapid detection of Klebsiella pneumoniae by magnetic immunocapture-loop-mediated isothermal amplification assay. Jundishapur J. Microbiol., 12: e90016.
Zhang, L; Wei, Q; Han, Q; Chen, Q; Tai, W; Zhang, J; Song, Y and Xia, X (2018). Detection of Shigella in milk and clinical samples by magnetic immunocaptured-loop-mediated isothermal amplification assay. Front. Microbiol., 9: 94.
Zhao, Y; Jiang, X; Qu, Y; Pan, R; Pang, X; Jiang, Y and Man, C (2017). Salmonella detection in powdered dairy products using a novel molecular tool. J. Dairy Sci., 100: 3480-3496.
Zheng, Q; Miks-Krajnik, M; Yang, Y; Lee, SM; Lee, SC and Yuk, HG (2016). Evaluation of real-time PCR coupled with immunomagnetic separation or centrifugation for the detection of healthy and sanitizer-injured Salmonella spp. on mung bean sprouts. Int. J. Food Microbiol., 222: 48-55.
Zhuang, L; Gong, J; Li, Q; Zhu, C; Yu, Y; Dou, X; Liu, X; Xu, B and Wang, C (2014). Detection of Salmonella spp. by a loop-mediated isothermal amplification (LAMP) method targeting bcfD gene. Lett. Appl. Microbiol., 59: 658-664.

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