CC BY
10DOI: https://doi.org/10.21323/2414-438X-2022-7-2-125-130 ©commons
Available online at https://www.meatjournal.ru/jour
Review article Open Access
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION A t d Receed
Accepted in revised 15.06.2022
(LAMP) METHOD FOR FAST DETECTION Accepted for publication 15.06.2022
OF CAMPYLOBACTER SPP IN MEAT FOOD PRODUCTS AND ENVIRONMENTAL OBJECTS OF A PROCESSING PLANT'S
Yuliya K. Yushina1,* Elena V. Zajko1, Maria A. Grudistova1, Maksim D. Reshchikov1, Nasarbay A. Nasyrov1, Dmitriy V. Nikitchenko2
1 V. M. Gorbatov Federal Research Center for Food Systems, Moscow, Russia 2 Peoples' Friendship University of Russia, Moscow, Russia
Keywords: bacteria of the genus Campylobacter, PCR, loop isothermal amplification method (LAMP), poultry and meat processing enterprises
Abstract
There is constant necessity of developing the accurate and fast methods for detection of foodborne pathogens. Microorganisms of Campylobacter genus are one of the main causes of foodborne diseases worldwide. Fast identification of Campylobacter at all stages of the food life cycle is an efficient strategy to control foodborne campylobacteriosis. This article the authors evaluated a commercial loop-mediated isothermal amplification (LAMP) system with bioluminescence, called as the 3M™ Molecular Detection Analysis (MDA), which was used to find Campylobacter in food products with the help of a certain standard method, which is referred to as the reference method. The results of this study showed that the commercial LAMP-based method is as efficient as the reference method, and features high specificity and minimum determinability (sensitivity). The LAMP-based method has been shown to be a fast and reliable method for detection of Campylobacter spp. scarce presence (10 CFU/25 g) in meat, meat products, as well as carcass swabs and production facilities' environment. The LAMP analysis required about 24-27 hours to achieve a result. However the LAMP-based method will facilitate the detection of Campylobacter, as it provides much easier and faster detection of Campylobacter spp., including Campylobacter jejuni/Campylobacter coli, than standard microbiological methods. The LAMP-based method is an efficient tool to prevent the spreading of Campylobacter spp. contamination in food products.
For citation: Yushina, Yu.K., Zajko, E.V., Grudistova, M.A., Reshchikov, M.D., Nasyrov, N.A. (2022). Loop-mediated isothermal amplification (LAMP) for fast detection of Campylobacter spp in meat food products and environmental objects of a processing plant Theory and Practice of Meat Processing, 7(2), 125-130. https://doi.org/10.21323/2414-438X-2022-7-2-125-130
Funding:
This work was supported by a grant from the Ministry of Science and Higher Education of the Russian Federation for large scientific projects in priority areas of scientific and technological development (grant number 075-15-2020-775).
Introduction
Campylobacteriosis is one of the main causes of bacterial diarrhea all over the world [1,2,3]. Two species of bacteria Campylobacter jejuni and Campylobacter coli are considered the main causes of campylobacteriosis in human [4]. Poultry products remain the main sources of pathogens which cause this disease. Organisms of the genus Campylobacter spp. are not usually pathogenic in adult poultry and are considered to be commensal microorganisms of the poultry's gastrointestinal microbiota. Therefore, campylobacteriosis spreads quickly and asymptomatically among the poultry and is extremely difficult to detect and trace [5]. According to the Center for Active Surveillance for Foodborne Diseases (FoodNet), 14 cases of campylobacteriosis are diagnosed per 100,000 population in the United States (USA) (approximately 1.3 million people) and 71 cases per 100,000 population in the European Union (EU) (approximately 200,000 people) every year [1,6]. Campylobacteriosis is rarely a fatal disease and rare reports of mortality are usually recorded
for the old age groups (elderly consumers) and/or immunocompromised patients (WHO).
The main target of activity to reduce the number of infections is the timely identification of Campylobacter spp. in food products.
In order to identify this pathogen the standard ISO 10272-1:2017 is used [7]. This standard provides for application of modified charcoal cefoperazone deoxycho-late agar (m CCDA). However, it has recently been found that Escherichia coli and Pseudomonas spp, which are widely distributed in poultry, also actively grow together with Campylobacter spp. under the same conditions when found in the same sample [8,9]. This is explained by acquired resistance of Escherichia coli and Pseudomonas spp. to selective factors of nutrient media recommended in the study [7]. This microorganisms' competition can lead to a significant underestimation of the presence of Campylo-bacter spp. in the tested matrix. Thus, there is a need to improve the methodology for detecting Campylobacter spp.
Copyright © 2022, Yushina et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons. org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
in food products. Traditional methods of microbiological cultivation must constantly evolve by expanding the scope of application of selective media and by optimizing the growth conditions to suppress the growth of associated microflora, including new antibiotics [10].
Although methods for isolating Campylobacter spp. continue to improve, problems remain that reduce the efficiency of these methodologies. When exposed to stress factors such as refrigeration and freezing, Campylobacter spp. can become viable but not culturable (VBNC), thus making it impossible to detect it by many traditional microbiological methods [11,12,13].
Bacteria in the VBNC state can remain dormant for several years and recover in favorable conditions and subsequently cause infections to human [14]. As a result infections and diseases outbreaks caused by VBNC bacteria are underestimated. It has been proven that 13 out of 20 chicken carcasses are likely to contain VBNC Campylobacter [15].
Fast PCR-based methods are to eliminate the problems and issues, associated with the certain difficulties of microbiological approaches, and allow fast assessment of degree of Campylobacter spp. incidence in various food matrices. Molecular methods for identifying and detecting food-borne pathogens are more sensitive. One of the most valuable methods, used in the food safety industry and science in general, is the polymerase chain reaction (PCR) [10].
The advanced and potential integrating systems such as LAMP-based microdevices, microchips, and other CD-LAMP-based systems are being developed today. These systems provide high specificity, high speed, multiple detection of foodborne pathogens and maximal minimum determinability (sensitivity). In addition, these LAMP-based systems provide the detection of opportunistic and pathogenic microorganisms [16].
The aim of this study is to evaluate the performance of a loop-mediated isothermal amplification (LAMP) based method with bioluminescence, called the 3M™ Molecular Detection System (MDS), used for fast detection of Campylobacter spp. in meat, including poultry meat samples and samples of the environmental objects and facilities.
Objects and methods
To determine the relative level of bacteria detection, the artificially contaminated samples of minced pork and minced chicken were used. Samples (minced pork and minced chicken) were purchased from a local supermarket in the central region of the Russian Federation in February-March, 2021.
To establish inclusion and exclusion of the pure cultures of 20 strains were used, including Campylobacter jejuni subsp. jejuni ATCC 8841 and Campylobacter coli ATCC 33559, and 18 Campylobacter spp. isolates, which were collected from poultry processing plants and previously found and confirmed by tests. On the other hand, exclusion tests were performed with non-target organisms using the 10 strains listed below in the Table 1.
Table 1. Microorganism strains used to define the specificity of the LAMP method
No Bacterial strains Source
1 Bacillus cereus ATCC 11778
2 Citrobacter freundii ATCC 43864
3 Enterococcus faecalis АТСС 29212
4 Escherichia coli АТСС 25922
5 Klebsiella pneumonia ATCC 13883, NCTC 9633
6 Proteus mirabilis ATCC 35659
7 Pseudomonas aeruginosa АТСС 27853
„ Salmonella enterica subsp. enterica a-t^^ ,
8 _ , . f ATCC 14028 serovar Typhimurium
9 Listeria innocua serotype 4a ATCC 33090, NCTC 11288
10 Salmonella enterica subsp. enterica 11272
serovar Enteritidis
Environmental swabs were analyzed to evaluate this method as a tool for monitoring the degree of Campylo-bacter spp. incidence. The swabs were taken at the poultry and meat processing plants in the Central region of Russia.
Definition of inclusion and exclusion
Campylobacter spp. strains were restored in Bolton broth (Merck, Germany), with further reinoculation on blood agar (OOO Sredoff, Russia) at temperature 41.5 °C for 48 hours under microaerophilic conditions (5% O2, 10% CO2, 85% N2). Microaerophilic conditions were created in the device Anaxomat (Advanced Instruments Inc., USA). Non-target microorganisms were grown on tryptic soy broth (TSB, Merck, Germany) at temperature 37 °C overnight under aerobic conditions, followed by its reinoculation on trypticase soy agar (TSA, Merck, Germany). Each test was performed five repetitions once — i. e. five replications. Then, strains of microorganisms of Campylobacter genus were introduced into Bolton broth (Merck, Germany) at a concentration of 100 CFU/225 ml, and non-target strains were inoculated in TSB (Merck, Germany) at a concentration of 1,000 CFU/225 ml. Further studies were carried out according to the method [7] and by the LAMP method, starting from the stage of enrichment.
The relative level of detection of Campylobacter spp. the LAMP method used in samples of minced chicken and minced pork was determined using bacterial cells of Campylobacter jejuni subsp. jejuni ATCC 8841 (from the American Type Culture Collection, Manassas, Virginia, USA). To prove the absence of Campylobacter in the food matrix (minced chicken and minced pork) used for further studies, the samples were examined according to ISO 10272-1:2017 [7] and the protocol of the LAMP method. Campylobacter-negative food matrices were used as a negative control sample in the analysis to establish the relative level of bacteria detection.
To prepare the required number of cells, Campylo-bacter spp. genus microorganisms were restored in Bolton
broth (Merck, Germany). Then they were centrifuged in a MiniSpin centrifuge (Eppendorf, Germany) at 3,000 g for 10 min, flushed twice in 0.85% sodium chloride solution (JSC VEKTON, Russia). The obtained cells were diluted by decimal dilutions, and the concentrations of microorganisms of 101 and 102 CFU/ml were obtained. Before inoculation, the amount of the prepared Campylobacter coli suspension was counted by spreading a 100 |l aliquot on mCCD agar (Merck, Germany) in duplicate; and the suspension was incubated at 42 °C for 48 hours under micro-aerophilic conditions to assess the level of inoculum. After confirming the required number of cells, the minced meat samples were inoculated. There were two inoculation levels for the matrices: the high inoculation level of approximately 100 CFU/25 g and the low inoculation level of approximately 10 CFU/25 g. After inoculation the samples were tested by two methods to reveal the presence of Campylobacter spp.
Campylobacter spp. was detected using a commercial LAMP-based kit (3M Molecular Detection Analysis 2 — Campylobacter; 3M). The strain was detected according to the manufacturer's guidelines. 25 g sample was mixed with 225 ml 3M Enrichment broth (3M, USA). Then 20 |l of 3M Enrichment broth was added to the vial with lysis solution. The mixture was heated in a thermoblock (Germany, IKA) at 100 °C for 15 min followed by its immediate cooling at room temperature in a cooling block (3M, USA) for 10 min. After cooling, 20 |l of the lysate was mixed with the lyophilized mixture in the test vial with the reagent included in the kit. The prepared vial with the reagent was put into a molecular detection system (3M, USA) for the detection of Campylobacter spp. cells using isothermal amplification and bioluminescence for 75 min. All analyses included negative controls and reagent controls to test the performance of the molecular detection system.
To test for the presence of Campylobacter spp. in accordance with ISO 10272-1:2017 [7], 25 g of the sample was homogenized in 225 ml of Bolton broth (Merck, Germany) and incubated under microaerophilic conditions for 4-6 hours at temperature 37 °C, and then 40-48 hours at temperature 41.5 °C. After the end of incubation the enriched material from the broth was streaked onto mCCD agar (Merck, Germany) and Preston selective agar (Oxoid, United Kingdom) with a 3 mm loop, and incubated under microaerophilic conditions at 41.5 °C for 44±4 hours. The colonies typical of Campylobacter spp. were inoculated on blood agar and then confirmed with biochemical tests api® Campy (bioMerieux, France).
Results and discussion
Microorganisms of the genus Campylobacter spp. are responsible for approximately 17% of human diarrhea worldwide, what makes them one of the leading causes of foodborne gastrointestinal infections. Governments and industry in many countries develop strategies to reduce the level of food contamination with Campylobacter spp.
[17]. There is an urgent need for fast and simple methods capable to detect Campylobacter spp. throughout the entire chain of food production. At this stage of the study, the efficiency of the LAMP method (using a commercial kit) was evaluated in comparison with the method according to ISO 10272-1:2017 [7].
The specificity of the method was assessed in terms of inclusion and exclusion. Twenty bacterial strains (Cam-pylobacter jejuni subsp. jejuni ATCC 8841 and Campylo-bacter coli ATCC 33559, 18 Campylobacter spp. isolates) were tested for inclusion. During the LAMP method testing, the positive results were obtained only with samples containing Campylobacter spp., including Campylobacter jejuni and Campylobacter coli, while no amplification was found in experiments with ten non-target strains of microorganisms (Table 2). In a parallel study according to ISO 10272-1:2017 [7] only target strains of microorganisms were identified and validated.
Table 2. Results of inclusion and exclusion of the LAMP analysis for Campylobacter spp.
NO. of tests
Strains LAMP ISO 10272-1:2017
Posit. Negat. Posit. Negat. Results of inclusivity
Campylobacterjejuni s^p. 5/5* 0/5 5/5 0/5 jejuni ATCC 8841
Campylobacter coli ATCC 33559 5/5* 0/5 5/5 0/5
Campylobacter spp. 18/18 0/18 18/18 0/18
Results of exclusivity Untargeted strains** 0/10 10/10 0/10 10/10
Notes:
* number of the test replications; ** referred to in the chapter "Objects".
The data in the Table 2 above show that the LAMP method was highly specific for Campylobacter spp., including Campylobacter coli and Campylobacter jejuni subsp. je-juni. The specificity of the method was 100%. The results obtained are also confirmed by a number of specialists' studies, which proved the high specificity of LAMP amplification, since 4 primers should specifically bind to 6 separate sections of the target DNA [18].
To determine the minimum determinability (sensitivity) of the LAMP method, various concentrations of the target Campylobacter jejuni ATCC 8841 were tested in the artificially contaminated samples of minced meat (Table 3).
At the levels of inoculation of 10 CFU/25 g and 100 CFU/25 g, the method could detect Campylobacter in all samples (sensitivity amounted to 100%), and the result in all samples was confirmed by the reference method. In a similar study, the detection limit for Campylobacter spp. by the LAMP method accounted for 1,000 CFU/g after incubation and 1-5 CFU/sample before enrichment. At the same time, the minimum determinability (sensitivity) of the reference method ISO 10272-1:2017 [7] was lower than
that of the LAMP method, which was consistent with the study by Rajagopal et al. [19].
Table 3. The results of the test for the minimum determinability (sensitivity) of the LAMP method for Campylobacter spp. in the artificially contaminated samples
Level of inoculation (CFU/g) Minced meat samples ISO 10272-1:2017 / LAMP Minimum determinability (sensitivity) (%) ? £ ci
T pos T neg F pos F neg ci V Oi c«
Poultry 0/5 5/5 0/5 0/5 100 100
100 (not inoculated) Pork 0/5 5/5 0/5 0/5 100 100
101 Poultry 5/5 0/5 0/5 0/5 100 100
Pork 5/5 0/5 0/5 0/5 100 100
102
Poultry 5/5 0/5 0/5 0/5 100 100 Pork 5/5 0/5 0/5 0/5 100 100
A significant number of studies have reported that the minimum determinability (sensitivity) of PCR methods is often higher than the one of classical microbiological methods [20, 21]. When examining a non-inoculated sample (the negative control sample), both methods did not detect Campylobacter spp. No false negative results were obtained.
To confirm the sensitivity and performance of the LAMP method, the researchers monitored the rate of Campylobacter spp. detection in the objects of the production plant's environment (poultry processing plant and meat processing plant). In total 308 samples were collected and tested (Table 4).
Table 4. Minimum determinability (sensitivity) results of the LAMP method compared to the method ISO 10272-1:2017, used for detection of Campylobacter spp. in the samples of native contamination
Samples (swabs) LAMP / ISO 10272-1:2017 Minimum determinability (sensitivity) (%) 2 £ ci
T T F F pos neg pos neg 5 Oi S
Meat processing plant 14/152 138/152 14/152 138/152 100 100
Poultry processing plant 60/156 96/156 57/156 99/156 100 94
Note:
T , T are true-positive and true-negative samples confirmed by
pos neg * or J
both ISO 10272-1:2017 [7] and LAMP methods; F and F are false-
pos neg
positive and false-negative samples confirmed only by the LAMP method or the ISO 10272-1:2017 method [7], respectively; * after the slash, the number of samples tested at the corresponding level of contamination is shown.
During the analysis by the LAMP method, 14 positive samples were found of 152 samples of the environment in the meat processing plant; and 60 positive samples were found of 156 samples of the environment in the poultry processing plant. All positive samples, obtained by
LAMP method, were confirmed according to the method ISO 10272-1:2017 [7] starting from their reinoculation from enrichment media to solified media. 3 samples (swabs) were taken from a poultry processing plant, that proved to be positive for Campylobacter spp. when using the LAMP method, however they gave a negative result when analyzed by traditional methods. The discrepancy in results may have been caused by the low number of Cam-pylobacter spp. in the enrichment media, which was insufficient for their detection by the conventional culturing methods. Another reason could be the increased antimicrobial resistance in representatives of Enterobacteriaceae in recent years. The problem of Campylobacter detection in poultry meat samples is confirmed by a wide number of publications [22, 23, 24, 25]. It was shown that ^-lactamase-resistant Escherichia coli, that is widespread in poultry, significantly outgrow Campylobacter spp. when both organisms are present in the same sample [8]. In addition, not only E. coli, but also other bacteria such as Pseudomonas spp. were found in large numbers in poultry samples and poultry processing plant's environment, and it may impede the detection of Campylobacter spp. [9].
The group of researchers Sabike et al. [26] evaluated the Loop-mediated Isothermal Amplification (LAMP) method, applied for direct screening of naturally contaminated chickens' cloacal swabs for Campylobacter jejuni/ Campylobacter coli to compare this analysis with conventional quantitative microbiological methods. In the comparative study LAMP analysis of 165 broilers showed a sensitivity of 82.8% (48/58 in conventional culturing), 100% (107/107) specificity, 100% (48/48) positive predictive value, and 91.5%. (107/117) negative predictive value. LAMP analysis took less than 90 minutes from the moment of faecal samples obtaining till getting the final results from the laboratory. This suggests that LAMP analysis would facilitate the identification of Campylobacter jejuni/Campylobacter coli.-positive broiler flocks at the farm or at slaughterhouses prior to slaughter, making it an efficient way to prevent the spread of Campylobacter infection [26].
In the course of this study, it was noted that the LAMP method showed high specificity with a significantly shorter time of analysis: 24-27 hours in comparison with 5-7 days of analysis by the conventional control method of culturing.
It is reported that the sensitivity of PCR methods is often higher than that of classical microbiological methods [20,21]. In another study, an optimized LAMP analysis was tested on the cells of Campylobacter jejuni and Campylo-bacter coli inoculated into the samples of chicken faeces. High sensitivity has also been demonstrated in the LAMP analysis, where LOD of 1 CFU/reaction (corresponding to 50 CFU/mL) was observed for both Campylobacter jejuni and Campylobacter coli within 30 minutes of amplification [4]. This suggests that LAMP analysis would facilitate the identification of C at the farm or at pre-slaughter stage in
the slaughterhouses, thus making it an efficient means of preventing the spread of Campylobacter spp.
Detection of foodborne pathogens using traditional culturing methods is a reliable approach, but to obtain the results it is necessary to use the specialized laboratories and wait for several days, so there is no real-time information on pathogens presence or absence [27]. Molecular analysis based on LAMP is used to detect a wide range of pathogens [28,29].
Fresh chicken meat suspected of being contaminated with Campylobacter jejuni / Campylobacter coli was subjected to analysis. For these samples, analysis takes approximately 24-48 hours from the start of the cumulative phase till the pathogen was finally detected. The sensitivity of the LAMP analysis has been found to be ten times higher than the sensitivity of the equivalent PCR analysis. The increase of LAMP sensitivity can be proven both by turbidimetric analysis and by visual assessment with the naked eye. The LAMP analysis is faster and easier to perform than conventional PCR analyses, and is also more specific and requires only a simple heating block or constant temperature water bath [30]. The development of molecular methods, including the LAMP-based methods, is currently receiving a lot of attention. In the research of Kreitlow et al., the developed LAMP analysis system based on the rplD and cdtC-gyrA genes provided a fast, sensitive, and highly specific method for detecting and differentiating Campylobacter jejuni and Campylobacter coli in meat products [31].
Conclusion
This study represents data of validation the performance of a new fast method for detection of Campylobacter in poultry carcasses compared to conventional methods of culturing based on the standard ISO 10272-1:2017.
Thus, the present study describes a simple and fast LAMP analysis used for the detection of Campylobacter jejuni and Campylobacter coli in the analyzed samples. Validation of the LAMP method in comparison with the standard method of mCCDA coating showed that LAMP has a certain limit of detection (LOD) of 10 CFU/25 g. This method features good specificity as all strains of Campylo-bacter jejuni and Campylobacter coli were detected without failure, and no false positive results were obtained in analyses of the other tested bacterial control strains. These parameters were determined by analyzing 152 environmental samples obtained from the pork and poultry processing plant, as well as naturally contaminated pork and poultry carcasses. Only 3 samples that were found Campylobacter positive by conventional LAMP methods, turned out to be culture negative, ie. false negative. To validate the alternative method, all positive results were validated by the standard method ISO 10272-1:2017.
LAMP method and the reference method showed very good compliance between them. It was evident that the LAMP method was very specific with a much shorter time of analysis: 24-27 hours compared to 5-7 days of the reference method of culturing. The performance of the LAMP method was equivalent to the reference method.
REFERENCES
1. Centers for Disease Control and Prevention (CDC) (2017). Foodborne Diseases Active Surveillance Network (FoodNet): Food-Net 2015 Surveillance Report (Final Data). Atlanta: U. S. Department of Health and Human Services. Retrieved from https://www. cdc.gov/foodnet/reports/annual-reports-2015.html. Accessed April 22, 2022.
2. Centers for Disease Control and Prevention (CDC) (2021). Cam-pylobacter (Campylobacteriosis). Retrieved from https://www.cdc. gov/foodsafety/diseases/campylobacter. Accessed April 22, 2022.
3. Helwigh, B., Christensen, J., Müller, L. (2016). Annual Report on Zoonoses in Denmark 2016. Soborg: National Food Institute, 2016.
4. Quyen, T. L., Nordentoft, S., Vinayaka, A. C., Ngo, T. A., En-gelsmenn, P., Sun, Y. et al. (2019). A sensitive, specific and simple loop mediated isothermal amplification method for rapid detection of campylobacter spp. in broiler production. Frontiers in Microbiology, 10(0CT), Article 2443. https://doi.org/10.3389/ fmicb.2019.02443
5. Horrocks, S. M., Anderson, R. C., Nisbet, D. J., Ricke, S. C. (2009). Incidence and ecology of Campylobacter jejuni and coli in animals. Anaerobe, 15(1-2), 18-25. https://doi.org/10.1016/j. anaerobe.2008.09.001
6. EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control). (2017). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2016. EFSA Journal, 15(12), 5077. https://doi.org/10.2903/j.efsa.2017.5077
7. ISO 10272-1:2017 Microbiology of the food chain — Horizontal method for detection and enumeration of Campylobacter spp. — Part 1: Detection method. Retrieved from https://www. iso.org/standard/63225.html. Accessed April 22, 2022.
8. Jasson, V., Sampers, I., Botteldoorn, N., Lopez-Galvez, F., Baert, L., Denayer, S. et al. (2009). Characterization of Escherich-ia coli from raw poultry in Belgium and impact on the detection of Campylobacter jejuni using Bolton broth. International Journal of
Food Microbiology, 135(3), 248-253. https://doi.Org/10.1016/j. ijfoodmicro.2009.09.007
9. Baylis, C. L., MacPhee, S., Martin, K. W., Humphrey, T. J., Betts, R. P. (2000). Comparison of three enrichment media for the isolation of Campylobacter spp. from foods. Journal of Applied Microbiology, 89(5), 884-891. https://doi.org/10.1046/j.1365-2672.2000.01203.x
10. Ricke, S. C., Feye, K. M., Chaney, W. E., Shi, Z., Pavlidis, H., Yang, Y. (2019). Developments in rapid detection methods for the detection of foodborne campylobacter in the United States. Frontiers in Microbiology, 10(JAN), Article 3280. https://doi. org/10.3389/fmicb.2018.03280
11. Tholozan, J. L., Cappelier, J. M., Tissier, J. P., Delattre, G., Federighi, M. (1999). Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells. Applied and Environmental Microbiology, 65(3), 1110-1116. https://doi. org/10.1128/aem.65.3.1110-1116.1999
12. Ziprin, R. L., Droleskey, R. E., Hume, M. E., Harvey, R. B. (2003). Failure of viable nonculturable Campylobacter jejuni to colonize the cecum of newly hatched leghorn chicks. Avian Diseases, 47(3), 753-758. https://doi.org/10.1637/7015
13. Castro, A. G., Dorneles, E. M., Santos, E. L., Alves, T. M., Silva, G. R., Figueiredo, T. C. et al. (2018). Viability of Campylobacter spp. in frozen and chilled broiler carcasses according to real-time PCR with propidium monoazide pretreatment. Poultry Science, 97(5), 1706-1711. https://doi.org/10.3382/ps/pey020
14. Oliver, J. D. (2005). The viable but nonculturable state in bacteria. Journal of Microbiology, 43, 93-100.
15. Duarte, A., Botteldoorn, N., Coucke, W., Denayer, S., Dierick, K., Uyttendaele, M. (2015). Effect of exposure to stress conditions on propidium monoazide (PMA)-qPCR based Campylobacter enumeration in broiler carcass rinses. Food Microbiology, 48, 182190. https://doi.org/10.1016/j.fm.2014.12.011
16. Lakshmi, B. A., Kim, S. (2021). Recent trends in the utilization of LAMP for the diagnosis of viruses, bacteria, and allergens
in food. Chapter in a book: Recent developments in applied microbiology and biochemistry. Academic Press, 2021. https://doi. org/10.1016/B978-0-12-821406-0.00027-8
17. Havelaar, A. H., Kirk, M. D., Torgerson, P. R., Gibb, H. J., Hald, T., Lake, R. J. et al. (2010). Group, on behalf of WHOFDBER (2015). World health organization global Estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Medicine, 12(12), Article e1001923. https://doi.org/10.1371/ journal.pmed.1001923
18. Li, Y., Fan, P., Zhou, S., Zhang, L. (2017). Loop-mediated isothermal amplification (LAMP): A novel rapid detection platform for pathogens. Microbial Pathogenesis, 107, 54-61. https://doi. org/10.1016/j.micpath.2017.03.016
19. Rajagopal, R., Barnes, C.A., David, J.M., Goseland, J. (2021). Evaluation of a commercial loop-mediated isothermal amplification assay, 3MTM Molecular Detection Assay 2 — Campylobacter, for the detection of Campylobacter from poultry matrices. British Poultry Science, 62(3)404-413. https://doi.org/10.1080/0007 1668.2021.1879992
20. Delpiazzo, R., Barcellos, M., Barros, S., Betancor, L., Fraga, M., Gil, J. et al. (2021). Accurate and fast identification of Campylobacter fetus in bulls by real-time PCR targeting a 16S rRNA gene sequence Veterinary and Animal Science, 11, Article 100163. https://doi.org/10.1016/j.vas.2020.100163
21. Linton, D., Owen, R.J., Stanley, J, (1996). Rapid identification by PCR of the genus Campylobacter and of five Campylobacter species enteropathogenic for man and animals. Research in Microbiology, 147(9), 707-718. https://doi.org/10.1016/s0923-2508(97)85118-2
22. Belmar Campos, C., Fenner, I., Wiese, N., Lensing, C., Christner, M., Rohde, H. at al. (2014). Prevalence and genotypes of extended spectrum beta-lactamases in Enterobacteriaceae isolated from human stool and chicken meat in Hamburg, Germany. International Journal of Medical Microbiology, 304(5-6), 678684. https://doi.org/10.1016/j.ijmm.2014.04.012
23. Cantdn, R., Novais, A., Valverde, A., Machado, E., Peixe, L., Baquero, F. et al. (2008). Prevalence and spread of extended-spectrum p-lactamase-producing Enterobacteriaceae in Europe. Clinical Microbiology and Infection, 14(SUPPL. 1), 144-153. https://doi.org/10.1111/j.1469-0691.2007.01850.x
24. Dierikx, C., van der Goot, J., Fabri, T., van Essen-Zandbergen, A., Smith, H., Mevius, D. (2013). Extended-spectrum-p-lactamase-and AmpC-p-lactamase-producing Escherichia coli in Dutch broilers and broiler farmers. Journal of Antimicrobial Chemotherapy, 68(1), 60-67. https://doi.org/10.1093/jac/dks349
25. Machado, E., Coque, T. M., Canton, R., Sousa, J. C., Peixe, L. (2008). Antibiotic resistance integrons and extended-spectrum p-lactamases among Enterobacteriaceae isolates recovered from chickens and swine in Portugal. Journal of Antimicrobial Chemotherapy, 62(2), 296-302. https://doi.org/10.1093/jac/dkn179
26. Sabike, I. I., Uemura, R., Kirino, Y., Mekata, H., Sekiguchi, S., Okabayashi, T. et al. (2016). Use of direct LAMP screening of broiler fecal samples for Campylobacter jejuni and Campylobacter coli in the positive flock identification strategy. Frontiers in Microbiology, 7(SEP), Article 1582. https://doi.org/10.3389/fmicb.2016.01582
27. Romero, M. R., Cook, N. (2018). A rapid LAMP-based method for screening poultry samples for Campylobacter without enrichment. Frontiers in Microbiology, 9(OCT), Article 2401. https://doi. org/10.3389/fmicb.2018.02401
28. Gunasegar, S., Neela, V. K. (2021). Evaluation of diagnostic accuracy of loop-mediated isothermal amplification method (LAMP) compared with polymerase chain reaction (PCR) for Leptospira spp. in clinical samples: a systematic review and meta-analysis. Diagnostic Microbiology and Infectious Disease, 100(3), Article 115369. https://doi.org/10.1016/j.diagmicrobio.2021.115369
29. Shang, Y., Ye, Q., Cai, S., Wu, Q., Pang, R., Yang, S. et al. (2021). Loop-mediated isothermal amplification (LAMP) for rapid detection of Salmonella in foods based on new molecular targets. LWT, 142, Article 110999. https://doi.org/10.1016/j.lwt.2021.110999
30. Mori, Y., Nagamine, K., Tomita, N., Notomi, T. (2001). Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochemical and Biophysical Research Communications, 289(1), 150-154. https://doi.org/10.1006/bbrc.2001.5921
31. Kreitlow, A., Becker, A., Ahmed, M. F., Kittler, S., Schotte, U., Plotz, M. et al. (2021). Combined loop-mediated isothermal amplification assays for rapid detection and one-step differentiation of Campylobacter jejuni and Campylobacter coli in meat products. Frontiers in Microbiology, 12, Article 668824. https://doi. org/10.3389/fmicb.2021.668824
AUTHOR INFORMATION
Yulia K. Yushina, Candidate of Technical Sciences, Docent, Deputy Head of Laboratory «Center for food and feed testing», V. M. Gorbatov Federal Research Center for Food Systems. 26, Talalikhina str., 109316, Moscow, Russia. Tel.: +7-495-676-91-26, E-mail: yu.yushina@fncps.ru ORCID: https://orcid.org/0000-0001-9265-5511 * corresponding author
Elena V. Zayko, Researcher, Department of Hygiene of Production and Microbiology, V. M. Gorbatov Federal Research Center for Food Systems. 26, Talalikhina str., 109316, Moscow, Russia, Tel.: +7-960-548-71-95, E-mail: el.zaiko@yandex.ru ORCID: https://orcid.org/0000-0002-5048-9321
Maria A. Grudistova, Researcher, Department of Hygiene of Production and Microbiology, V. M. Gorbatov Federal Research Center for Food Systems. 26, Talalikhina str., 109316, Moscow, Russia, Tel.: +7-916-311-64-30, E-mail: m.grudistova@fncps.ru ORCID: https://orcid.org/0000-0002-8581-2379
Maksim D. Reshchikov, Senior Laboratory Assistant, Department of Hygiene of Production and Microbiology, V. M. Gorbatov Federal Research Center for Food Systems. 26, Talalikhina str., 109316, Moscow, Russia, Tel.: +7-962-959-32-22, E-mail: m.reshchikov@fncps.ru ORCID: https://orcid.org/0000-0002-1344-823X
Nazarbay A. Nasyrov, Researcher, Department of Hygiene of Production and Microbiology, V. M. Gorbatov Federal Research Center for Food Systems. 26, Talalikhina str., 109316, Moscow, Russia, Tel.: +7-906-830-17-77, E-mail: n.nasyrov@fncps.ru ORCID: https://orcid.org/0000-0001-8545-6266
Dmitriy V. Nikitchenko, Doctor of Biological Sciences, Docent, Professor, Department of Veterinary Medicine, Peoples' Friendship University of Russia, 6, Miklukho-Maklaya str., 117198, Moscow, Russia, Tel.: +7-910-421-32-58, E-mail: dvnikitchenko@mail.ru ORCID: https://orcid.org/ 0000-0003-2294-6908
All authors bear responsibility for the work and presented data.
All authors made an equal contribution to the work.
The authors were equally involved in writing the manuscript and bear the equal responsibility for plagiarism. The authors declare no conflict of interest.
