Skip to main content
NCBI home page
Iranian Journal of Veterinary Research logoLink to Iranian Journal of Veterinary Research
. 2020 Winter;21(1):20–25.

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

L Zhang 1,**, X Du 1,**, C Chen 1, Q Han 2, Q Chen 2, M Zhang 2, X Xia 2, Y Song 2, J Zhang 2,*
PMCID: PMC7183372  PMID: 32368221

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.

Key Words: Detection, LAMP, Magnetic Immunocapture, Monoclonal antibody, Salmonella

Introduction

Salmonella is an important bacterial pathogen worldwide, infecting humans and a variety of animals. Infection can spread through habitats, feeding activities, or reservoirs (Zhang et al., 2003 ▶; Alves et al., 2015 ▶). Salmonella may cause several diseases including typhoid fever and salmonellosis, when it is ingested (Royer and Pare, 2003 ▶; Salzman et al., 2003 ▶). Salmonella infects approximately 94 million people globally each year, including 80.3 million food-borne cases and 155,000 deaths (Majowicz et al., 2010 ▶). In 2013, the Economic Research Service (ERS) estimated that the monetary costs of countering salmonellosis in the United States were over $3,666 million. Improved rapid and accurate detection methods are strongly needed. Culture-based technology was long considered the gold standard for detecting Salmonella in all kinds of samples. However, this process requires multiple subculture steps, and biochemical and serological confirmation is also needed. Together, these steps require about seven days, and is a time-consuming and labor-intensive process (Zhao et al., 2017 ▶). Loop-mediated isothermal amplification (LAMP) was described by Notomi et al. (2000) ▶. Loop-mediated isothermal amplification is a novel nucleic acid amplification method, that amplifies DNA with high specificity, sensitivity, rapidity and efficiency, and has been applied for the detection of various pathogens, including fungi, parasites, viruses, and bacteria (Mori and Notomi, 2009 ▶; Law et al., 2015 ▶; Song et al., 2016 ▶; Khan et al., 2018 ▶; Liu et al., 2018 ▶). A positive reaction generates white magnesium pyrophosphate precipitate, which can be easily observed by the naked eye, without the requirement of electrophoresis (Mori et al., 2001 ▶). This advantage allows the method to be performed as an on-site test using simple reaction equipment (Kanitkar et al., 2017 ▶). However, this method still has some limits, as it cannot enrich cells and it is typically necessary to enrich Salmonella before detection. Thus, it is difficult to apply this technology to various important samples, such as milk, seafood, and faeces.

In this study, LAMP was combined with magnetic immuno-capture technology in a process that showed a higher detection limit than that of polymerase chain reactions (PCR) and was more specific than the culture-based method. Use of the immunocapture-loop-mediated isothermal amplification (IC-LAMP) method for the detection of Salmonella was efficient, rapid, and showed high specificity.

Materials and Methods

Strains used in this study

All strains used in this study are shown in Supplementary Table (ST1). In addition, Shigella, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus were used for specificity testing.

Production of polyclonal antibody (pAb) and monoclonal antibody (mAb) against Salmonella

The production of pAb and mAb and against Salmonella was performed as was previously described (Zhang et al., 2019 ▶; Zhang et al., 2019 ▶). We use 107 CFU of Salmonella that was dissolved in phosphate buffer saline (PBS) and inactive for 1 h at 80°C as antigen to immunized mice (Zhang et al., 2018 ▶). The first immunization was performed using 107 CFU of Salmonella mixed with Freund’s complete adjuvant. The second and third immunizations were performed using 107 CFU of Salmonella mixed with Freund’s incomplete adjuvant. The booster immunization was performed using 107 CFU of Salmonella dissolved in PBS, after three days of the third immunization. Through three times normal immunizations and a booster immunization, the pAb were prepared. Then, the spleen cells of the mice were collected and fused with mouse myeloma cells (SP2/0) (Sun et al., 2015 ▶). The indirect enzyme-linked immunosorbent assay (ELISA) was used to determine the presence of Salmonella-specific antibodies in the hybridoma supernatants (Encheng et al., 2013 ▶; Du et al., 2016 ▶; Datta et al., 2018 ▶). The positive hybridomas will also be screened, after subcloning several times.

Purification and titration of Ascitic fluid

The female BALB/c mice (7-week-old) were used to produce ascites against Salmonella. Briefly, the mice were injected intraperitoneally with 0.5 ml of sterile paraffin oil. Seven days later, 106 cells of each hybridoma suspended in RPMI 1640 medium were used to inject each mouse. Ascitic fluid of mice was collected and purified using protein A affinity chromatography. The purified ascites were confirmed by Western blot and ELISA (Xia et al., 2016 ▶).

Western blot analysis

Western blot was used to identify the reactivity and specificity of ascites (Hemmatzadeh and Kazemimanesh, 2017 ▶). Briefly, the Salmonella were harvested and suspended in water and then inactivated. The resulting cellular lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, the proteins were transferred from the gel to a nitrocellulose (NC) membrane, and blocked with blocking buffer for 2 h at 37°C. The Ascitic fluid against Salmonella (1:2,000) were added and incubated for 2 h at 37°C. After incubation, the goat anti-mouse immunoglobulin G (IgG) heavy chain and light chain (H+L) (1:4,000; GenScript, USA) was used as the second antibody and was incubated for 1 h at 37°C. Finally, the NC membrane was scanned and analyzed with a chemiluminescence imaging system (BIOTOP Biosciences).

Genomic DNA extraction

All strains were grown on Luria-Bertani (LB) solid plates (Solarbio, China) first and then cultivated in LB liquid medium. The genomic DNA was extracted using a Bacteria Genomic DNA kit (Zomanbio, China), according to the manufacturer’s instructions.

The LAMP a ssay

The LAMP reaction was performed in 25 μL, containing a set of four primers (F3: 5´-CGC GGC CCG ATT TTC TCT-3´, B3: 5´-GGC AAT AGC GTC ACC TTT G-3´, FIP: 5´-GCG CGG CAT CCG CAT CAA TAT TTT GGT ATG CCC GGT AAA CAG AT-3´, and BIP: 5´- GCG AAC GGC GAA GCG TAC TGT TTT CAT CGC ACC GTC AAA GGA A-3´), 1 μL of Bacillus stearothermophilus (Bst) DNA polymerase (Eiken Chemical Co., Ltd., Tokyo, Japan), 2 μL of DNA template, 12.5 μL of 10 × LAMP buffer (Eiken Chemical Co., Ltd., Tokyo, Japan), 4 μL of dNTP mixture, and water. The mixture was maintained at 63°C for 40 min and terminated at 85°C for 5 min (Deb et al., 2017 ▶). The LAMP products were analyzed by separation on 3% agarose gel.

The standard IC-LAMP assay

The immuno-magnetic beads (IMBs) were prepared as described in our previous study (Zhang et al., 2018 ▶). After preparation of IMBs, they were used to capture Salmonella in different samples at 37°C for 0.5 h. Next, the remaining samples were removed and the IMBs were washed three times with phosphate buffer saline containing 0.05% Tween-20 (PBS-T). The enriched products (IMBs-Salmonella) were then lysed with 10 μL of solution A (125 mM NaOH, 1 mM EDTA, 0.1% Tween-20) and incubated for 10 min at 65°C. Finally, the reaction was terminated by addition of 10 μL of solution B (125 mM HCl, 10 mM Tris) (Zhang et al., 2019 ▶). Then, the LAMP reaction was performed as described above.

Specificity and sensitivity of LAMP, and IC-LAMP

Common foodborne pathogenic bacteria (Supplementary Table (ST1)) were cultured and used for the extraction of genomic DNA. Loop-mediated isothermal amplification and IC-LAMP assays were conducted to identify the specificity of the designed primers. Additionally, the sensitivity of the LAMP and IC-LAMP assays were verified using the different concentrations of Salmonella and plasmids.

Practical application of IC-LAMP to Salmonella detection in milk and water

To further verify the applicability of IC-LAMP technology, Salmonella was mixed with pasteurized milk samples and water. The Salmonella strains were then diluted into the milk and water samples for final concentrations of 5 × 107 to 5 × 100 CFU/ml. The milk and water samples were mixed with the IMBs in a tube and then incubated for 30 min at 37°C. The specificity of the IC-LAMP was determined in both processed milk and sterile water. To do this, bacterial mixtures were prepared with Salmonella (a mixture of Salmonella, E. coli, S. aureus, Shigella, P. aeruginosa, A. baumannii, and K. pneumoniae) or the bacterial mixtures without Salmonella. All strains were mixed together and diluted into skim milk or sterile water. The bacterial mixtures were captured with IMBs. The compounds of bread-cell were washed thoroughly and lysed as described above. Next, 2 μL of the bacteria lysate were used for IC-LAMP. Non-contaminated milk and water samples were used as negative controls.

Results

Characterization of Salmonella -specific mAbs

After the booster immunization, the sera of mice were collected and the titer of the pAb was tested by ELISA (Fig. 1A). Through three rounds of subcloning, two stable positive hybridomas were prepared and named as 1B12 and 2F1. Then, the hybridomas of 1B12 and 2F1 were used to produce ascites. The immunoglobulin isotypes of mAb 2F1 and 1B12 were determined by mouse mAb isotyping kit. The results indicated that mAb 1B12 and 2F1 were isotypes IgG3, and the light chains of the two mAbs belonged to the kappa chain family (Table 1). The titers of two mAbs were detected by ELISA (Fig. 1B). The concentrations of the two mAbs are shown in Table 1. ELISA analysis demonstrated that mAb 1B12 bound Salmonella specifically with high affinity (Kd = 1.126 ± 0.15 nM) (Fig. 1CA). The purity of the purified mAbs were confirmed by SDS-PAGE (Fig. 1D). The results of Western blot showed that mAb 1B12 only recognized Salmonella (Fig. 1E, Lanes 1 and 2) and showed no reactivity with the other similar bacteria.

Fig. 1.

Fig. 1

Open in a new tab

Characterization of anti-Salmonella pAb and mAb. (A) The titer of pAb, (B) The titer of mAb 1B12 and 2F1, (C) The saturation curve to determine the dissociation constants for mAb 1B12 (CA) and 2F1 (CB), (D) SDS-PAGE analysis of purified mAb 1B12 and 2F1. M: Protein marker, and (E) Western blot analysis of mAb 1B12. M: Protein marker. OD: Optical density

Table 1.

Characterization of the mAbs against Salmonella

mAbs Isotype mAb titer mAb concentration (mg/ml)
1B12 IgG3, κ chain 106 2.5
2F1 IgG3, κ chain 105 1.8
Open in a new tab

Specificity and sensitivity of LAMP

To measure the specificity, we tested the ability of the LAMP assay to generate signals from other bacterial samples. Based on the invA, two pairs of primers were designed successfully and could specifically amplify Salmonella, but no fragments were amplified from the other bacterial strains (Fig. 2A). To evaluate the sensitivity of LAMP, the plasmids were serially diluted from 109 to 100 copies/μL. The results of the LAMP showed that the trapezoidal strip was clearly observed even at a concentration of 100 copies/μL (Fig. 2B).

Fig. 2.

Fig. 2

Open in a new tab

Specificity and sensitivity of the LAMP assay against Salmonella or the plasmid containing the Salmonella invA gene. (A) Specificity of the invA-LAMP assay. M: 2000 bp DNA marker; 1: Salmonella (isolated from monkey); 2: Salmonella (ATCC8739); 3: Escherichia coli; 4: Staphylococcus aureus; 5: Klebsiella pneumoniae; 6: Acinetobacter baumannii; 7: Pseudomonas aeruginos; 8: Shigella; 9: Blank control, and (B) Sensitivity of the invA-LAMP assay (8 × 109 to 8 × 100 copies). M: 2000 bp DNA marker, and BC: Blank control

Specificity and sensitivity of IC-LAMP

Staphylococcus aureus, E. coli, Shigella, P. aeruginosa, Salmonella, A. baumannii, and K. pneumoniae were captured by IMBs. The sample of mixed bacteria lacking Salmonella was also captured by IMBs and the enriched products were used as a template to determine the specificity of the assay. The results showed that the trapezoidal strips clearly appeared when the mixed bacteria contained Salmonella (Fig. 3A). Next, the sensitivity of the IC-LAMP assay was tested with Salmonella serially diluted into sterile water (from 5 × 106 to 5 × 100 CFU/ml). The results showed that the trapezoidal strip was clearly observed even at a concentration of 5 × 100 CFU/ml (Fig. 3B).

Fig. 3.

Fig. 3

Open in a new tab

Specificity and sensitivity of the IC-LAMP assay tested for artificially contaminated water captured by IMBs. (A) Specificity of the IC-LAMP assay. M: 2000 bp DNA marker; 1: Salmonella (isolated from monkey); 2: Salmonella (ATCC8739); 3: Escherichia coli; 4: Staphylococcus aureus; 5: Klebsiella pneumoniae; 6: Acinetobacter baumannii; 7: Pseudomonas aeruginos; 8: Shigella; 9: Blank control, and (B) Sensitivity of the IC-LAMP assay (5 × 106 to 5 × 100 CFU/ml). M: 2000 bp DNA marker, and BC: Blank control

Detection of Salmonella contamination of milk using the IC-LAMP assay

The practical application of IC-LAMP was confirmed using artificial contamination of milk. The green fluorescence and trapezoidal bands were obviously observed when the milk samples contained mixed bacteria including Salmonella (Figs. 4A, C). Next, Salmonella were serially diluted into skim milk (from 5 × 105 to 5 × 100 CFU/ml) to assess the sensitivity of the IC-LAMP assay. Results showed that the amplified fragments and the green fluorescence were clearly observed even at a concentration of 5 × 100 CFU/ml (Figs. 4B, D).

Fig. 4.

Fig. 4

Open in a new tab

Specificity and sensitivity of the IC-LAMP assay tested by artificial contamination of milk captured by IMBs. (A) Specificity of the IC-LAMP assay. M: 2000 bp DNA marker; 1: Salmonella (isolated from monkey); 2: Salmonella (ATCC8739); 3: Escherichia coli; 4: Staphylococcus aureus; 5: Klebsiella pneumoniae; 6: Acinetobacter baumannii; 7: Pseudomonas aeruginos; 8: Shigella; 9: Blank control, (B) Sensitivity of the IC-LAMP assay (5 × 105 to 5 × 100 CFU/ml). M: 2000 bp DNA marker, and BC: Blank control, (C) Specificity of IC-LAMP assay based on SYBR Green I detection. 1: Salmonella (isolated from monkey); 2: Salmonella (ATCC8739); 3: Escherichia coli; 4: Staphylococcus aureus; 5: Klebsiella pneumoniae; 6: Acinetobacter baumannii; 7: Pseudomonas aeruginos; 8: Shigella, and (D) Sensitivity of the IC-LAMP assay based on SYBR Green I detection. 1-6: 5 × 105 to 5 × 100 CFU/ml, and 7: Blank control

Discussion

Presenting a significant food safety risk, Salmonella is a common cause of food poisoning worldwide (Handley et al., 2015 ▶; Karp et al., 2018 ▶). Culture-based techniques and biochemical assays are the most commonly used detection assays, but these methods require a long enrichment time and subsequent identification, and so cannot be used for rapid detection (Elzein et al., 2013 ▶; Soria et al., 2013 ▶). Polymerase chain reactions technologies can be used to assess whether various food products and environment samples contain Salmonella (Law et al., 2014 ▶; Singh et al., 2017 ▶). However, complex instruments are required to denature the template DNA and to detect the PCR products, greatly limiting the wide application of these technologies in under-resourced settings and in field laboratories (Hapuarachchi et al., 2019). Recently, the use of LAMP was reported as a tool to detect Salmonella (Shehata and Lu, 2016 ▶). Although these methods can detect Salmonella, there are still many shortcomings for this approach, because these methods cannot enrich cells, especially for some specialty samples.

In this study, we used Salmonella as the antigen to produce mAbs. Two mAbs were screened through three cycles of subcloning. The specificity of mAb 1B12 was evaluated by Western blot. The results of Western blot showed that mAb 1B12 bound Salmonella specifically and the other similar microbial strains including Shigella, E. coli, P. aeruginosa, K. pneumoniae, A. baumannii, and S. aureus were not recognized. We used mAb 1B12 with high affinity to develop and validate an IC-LAMP assay for the detection of Salmonella. This approach was rapid, sensitive, visual, and specific for the detection of Salmonella in contaminated milk and water. The overall detection requires about 50 min, including the enrichment of Salmonella. Moreover, the specificity and sensitivity of IC-LAMP reactions were successfully demonstrated. The IC-LAMP assay combined the specificity of antibody and LAMP exhibits a detection limit of 5 × 100 CFU/ml for processed milk and water samples and 100 copies/μL for plasmid, which is higher than direct PCR (103 CFU/ml) (Chin et al., 2017 ▶), real-time PCR coupled with immunomagnetic separation or centrifugation (105 CFU/ml for 30 min) (Zheng et al., 2016 ▶), and LAMP (5 CFU of Salmonella pure culture or 200 CFU of artificially spiked faeces) (Zhuang et al., 2014). Besides, compared with culture-based techniques and biochemical assays, PCR, real-time PCR, and normal LAMP, the IC-LAMP assay developed here only needed a portable heating apparatus without the processes of the enrichment of cells followed by extraction of the genome or plasmid, any special equipment, or professional skills.

In conclusion, this work demonstrates a reliable method to detect Salmonella based on LAMP combined with magnetic immunocapture assay IC-LAMP. The procedure is simple, rapid, and can be used for on-site detection of Salmonella.

Acknowledgements

This study was funded by the Yunnan Province Biomedical Science and Technology Plan Project (grant No. 2019ZF004-1), Applied Basic Research Projects of Yunnan Province (2018FB128), National Natural Science Foundation of China (grant No. 81860625).

Conflict of interest

The authors have no conflict of interest to declare.

References

  1. Alves J, Niguma NH, de Oliveira TCRM. Detection of Salmonella spp in eight complex food matrices using polymerase chain reaction assay. J. Food Safety. . 2015;35:453–457. [Google Scholar]
  2. Chin WH, Sun Y, Hogberg J, Quyen TL, Engelsmann P, Wolff A, Bang DD. Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples. Mol. Cell. Probes. 2017;32:24–32. doi: 10.1016/j.mcp.2016.11.004. [DOI] [PubMed] [Google Scholar]
  3. Datta S, Dandapat P, Jas R. Diagnosis of mixed gastrointestinal nematode infection in goat by an indirect-ELISA. Iran. J. Vet. Res. 2018;19:189–193. [PMC free article] [PubMed] [Google Scholar]
  4. Deb R, Sengar GS, Singh U, Kumar S, Raja TV, Alex R, Alyethodi RR, Prakash B. LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples. Iran. J. Vet. Res. 2017;18:134–137. [PMC free article] [PubMed] [Google Scholar]
  5. Du W, Wang Y, Huang L, Wei Y, Chen D, Sun J, Wu H, Feng L, Liu C. Characterization of monoclonal antibodies that recognize the amino- and carboxy-terminal epitopes of the pseudorabies virus UL42 protein. Appl. Microbiol. Biotechnol. 2016;100:181–192. doi: 10.1007/s00253-015-6957-7. [DOI] [PubMed] [Google Scholar]
  6. Elzein M, Alum A, Abbaszadegan M. Biochemical signature assay for use in a biosensor platform to detect bacteria in drinking water biofilms. Environ. Lett. 2013;48:925–932. doi: 10.1080/10934529.2013.762736. [DOI] [PubMed] [Google Scholar]
  7. Encheng S, Jing Z, Tao Y, Qingyuan X, Yongli Q, Wenshi W, Peng W, Liang S, Jing S, Donglai W. Analysis of murine B-cell epitopes on Eastern equine encephalitis virus glycoprotein E2. Appl. Microbiol. Biotechnol. 2013;97:6359–6372. doi: 10.1007/s00253-013-4819-8. [DOI] [PubMed] [Google Scholar]
  8. Handley JA, Shi Z, Si HP, Dawoud TM, Kwon YM, Ricke SC. Salmonella and the potential role for methods to develop microbial process indicators on chicken carcasses. Food safety. 2015;PP:81–104. [Google Scholar]
  9. Hapuarachchi CT, Jeffery KJM, Bowler ICJW. Stool PCR may not be a substitute for enrichment culture for the detection of Salmonella. J. Med. Microbiol. 2019;68:395–397. doi: 10.1099/jmm.0.000923. [DOI] [PubMed] [Google Scholar]
  10. Hemmatzadeh F, Kazemimanesh M. Detection of specific antigens of Newcastle disease virus using an absorbed western blotting method. Iran. J. Vet. Res. 2017;18:92–96. [PMC free article] [PubMed] [Google Scholar]
  11. Kanitkar YH, Stedtfeld RD, Hatzinger PB, Hashsham SA, Cupples AM. Development and application of a rapid, user-friendly, and inexpensive method to detect Dehalococcoides sp reductive dehalogenase genes from ground water. Appl. Microbiol. Biotechnol. 2017;101:4827–4835. doi: 10.1007/s00253-017-8203-y. [DOI] [PubMed] [Google Scholar]
  12. Karp BE, Campbell D, Chen JC, Folster JP, Friedman CR. Plasmid-mediated quinolone resistance in human non-typhoidal Salmonella infections: an emerging public health problem in the United States. Zoonoses. Public. Hlth. 2018;65:838–849. doi: 10.1111/zph.12507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Khan RSA, Ali W, Kiran S, Shah MSD, Tahir ZA, Habib M. Rapid detection of infectious bursal disease by loop-mediated isothermal amplification for field analysis. Iran. J. Vet. Res. 2018;19:101–107. [PMC free article] [PubMed] [Google Scholar]
  14. Law JW, Ab Mutalib NS, Chan KG, Lee LHM. Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations. Front. Microbiol. 2014;5:770. doi: 10.3389/fmicb.2014.00770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Liu S, Geng Y, Liu L, Sun X, Shao J, Han B, Wang J, Tan K. Development of an isothermal amplification-based assay for the rapid detection of Cronobacter spp. J. Dairy Sci. 2018;101:4914–4922. doi: 10.3168/jds.2017-13931. [DOI] [PubMed] [Google Scholar]
  16. Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O’Brien SJ, Jones TF, Fazil A, Hoekstra RM. The global burden of nontyphoidal Salmonella gastroenteritis. Clin. Infect. Dis. 2010;50:882–889. doi: 10.1086/650733. [DOI] [PubMed] [Google Scholar]
  17. Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 2001;289:150–154. doi: 10.1006/bbrc.2001.5921. [DOI] [PubMed] [Google Scholar]
  18. Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J. Infect. Chemother. 2009;15:62–69. doi: 10.1007/s10156-009-0669-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplification of DNA. Nucleic Acids. Res. 2000;28:E63. doi: 10.1093/nar/28.12.e63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Royer S, Pare D. Conservation of total synaptic weight through balanced synaptic depression and potentiation. Nature. . 2003;422:518–522. doi: 10.1038/nature01530. [DOI] [PubMed] [Google Scholar]
  21. Salzman NH, Ghosh D, Huttner KM, Paterson Y, Bevins CL. Protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin. Nature. . 2003;422:522–526. doi: 10.1038/nature01520. [DOI] [PubMed] [Google Scholar]
  22. Shehata DM, Lu X. Development of a loop mediated isothermal amplification (LAMP) - surface enhanced raman spectroscopy (SERS) assay for the detection of Salmonellaenterica serotype enteritidis. Theranostics. . 2016;6:522. doi: 10.7150/thno.14391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Singh G, Sithebe A, Enitan AM, Kumari S, Bux F, Stenstrom TA. Comparison of droplet digital PCR and quantitative PCR for the detection of Salmonella and its application for river sediments. J. Water Health. . 2017;15:505–508. doi: 10.2166/wh.2017.259. [DOI] [PubMed] [Google Scholar]
  24. Song J, Mauk MG, Hackett BA, Cherry S, Bau HH, Liu C. Instrument-free point-of-care molecular detection of Zika virus. Anal. Chem. 2016;88:7289–7294. doi: 10.1021/acs.analchem.6b01632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Soria MC, Soria MA, Bueno DJ, Terzolo HR. Comparison of 3 culture methods and PCR assays for Salmonella gallinarum and Salmonella pullorum detection in poultry feed. Poult. Sci. 2013;92:1505–1515. doi: 10.3382/ps.2012-02926. [DOI] [PubMed] [Google Scholar]
  26. Sun J, Huang L, Wei Y, Wang Y, Chen D, Du W, Wu H, Feng L, Liu C. Identification of three PPV1 VP2 protein-specific B cell linear epitopes using monoclonal antibodies against baculovirus-expressed recombinant VP2 protein. Appl. Microbiol. Biotechnol. 2015;99:9025–9036. doi: 10.1007/s00253-015-6790-z. [DOI] [PubMed] [Google Scholar]
  27. Xia T, Jia C, Ying W, Wang X. Gene cloning, expression and polyclonal antibody preparation of Rab3A for protein interaction analysis. Springer plus. 2016;5:1705. doi: 10.1186/s40064-016-3330-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zhang L, Du X, Chen C, Chen Z, Gong N, Song Y, Song Y, Han Q, Xia X, Luo H, Zhang J. Instrument-Free and Visual Detection of Salmonella Basedon Magnetic Nanoparticles and an Antibody Probe Immunosensor. Int. J. Mol. Sci. 2019;20(18) doi: 10.3390/ijms20184645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zhang S, Kingsley RA, Santos RL, Andrews-Polymenis H, Raffatellu M, Figueiredo J, Nunes J, Tsolis RM, Adams LG, Baumler AJ. Molecular pathogenesis of Salmonellaenterica serotype Typhimurium-induced diarrhea. Infect. Immun. 2003;71:1–12. doi: 10.1128/IAI.71.1.1-12.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zhang L, Shi Y, Chen C, Han Q, Chen Q, Xia X, Song Y, Zhang J. Rapid, visual detection of Klebsiella pneumoniae using magnetic nanoparticles and an horseradish peroxidase-probe based immunosensor. J. Biomed. Nanotechnol. 2019;15:1061–1071. doi: 10.1166/jbn.2019.2736. [DOI] [PubMed] [Google Scholar]
  31. Zhang L, Shi Y, Chen C, Han Q, Zhang M, Yi H, Song Y, Xia X, Zhang J. Visual and rapid detection of Klebsiella pneumoniae by magnetic immunocapture-loop-mediated isothermal amplification assay. Jundishapur J. Microbiol. 2019;12:e90016. [Google Scholar]
  32. Zhang L, Wei Q, Han Q, Chen Q, Tai W, Zhang J, Song Y, Xia X. Detection of Shigella in milk and clinical samples by magnetic immunocaptured-loop-mediated isothermal amplification assay. Front. Microbiol. 2018;9:94. doi: 10.3389/fmicb.2018.00094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zhao Y, Jiang X, Qu Y, Pan R, Pang X, Jiang Y, Man C. Salmonella detection in powdered dairy products using a novel molecular tool. J. Dairy Sci. 2017;100:3480–3496. doi: 10.3168/jds.2016-12535. [DOI] [PubMed] [Google Scholar]
  34. Zheng Q, Miks-Krajnik M, Yang Y, Lee SM, Lee SC, Yuk HG. 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. 2016;222:48–55. doi: 10.1016/j.ijfoodmicro.2016.01.013. [DOI] [PubMed] [Google Scholar]
  35. Zhuang L, Gong J, Li Q, Zhu C, Yu Y, Dou X, Liu X, Xu B, Wang C. Detection of Salmonella spp by a loop-mediated isothermal amplification (LAMP) method targeting bcfD gene. Lett. Appl. Microbiol. 2014;59:658–664. doi: 10.1111/lam.12328. [DOI] [PubMed] [Google Scholar]

Articles from Iranian Journal of Veterinary Research are provided here courtesy of Shiraz University

RESOURCES