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Application of loop-mediated isothermal amplification for detection of Yersinia enterocolitica in pork meat
Journal of Microbiological Methods 77 (2009) 198–201
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Application of loop-mediated isothermal amplification for detection of Yersinia enterocolitica in pork meat Hongwei Gao a,⁎, Zhiwen Lei a, Juntao Jia a, Shufeng Wang a, Ying Chen b, Min Sun a, Chengzhu Liang a a b
Shandong Entry–Exit Inspection and Quarantine Bureau of the People's Republic of China, Qingdao, China Chinese Academy of Inspection and Quarantine, Institute of Food Safety, Beijing, China
a r t i c l e
i n f o
Article history: Received 13 October 2008 Received in revised form 3 February 2009 Accepted 3 February 2009 Available online 12 February 2009 Keywords: Loop-mediated isothermal amplification Detection Yersinia enterocolitica
a b s t r a c t A loop-mediated isothermal amplification (LAMP) method was developed for the detection of Yersinia enterocolitica isolates in both pure bacterial cultures and pork meat. The LAMP primers, which corresponded to the gyrB gene, accurately identified 4 different bioserotypes of Y. enterocolitica. These primers failed to detect Y. pseudotuberculosis, Y. frederiksenii, and 17 non-Yersinia strains. The sensitivity of the LAMP assay for the detection of Y. enterocolitica in pure culture was 65 CFU/mL (31.6 fg of genomic DNA). The LAMP assay was conducted for the detection of Y. enterocolitica strains in 21 pig tonsil samples and 73 pork meat samples obtained from 94 slaughtered pigs belonging to 4 different herds. Y. enterocolitica was found to be present in 4 tonsil samples and none in meat samples. This is the first report in which the LAMP assay was employed for the detection of Y. enterocolitica in food samples. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Yersinia enterocolitica is a common enteric pathogen in humans. This pathogen has a strong propensity to cause acute gastroenteritis, enterocolitis, and mesenteric adenitis, as well as a variety of extraintestinal disorders (Bottone, 1999). This organism has been classified into approximately 60 serotypes. However, most strains that are associated with human yersiniosis belong to the bioserotypes 1B/O:8, 2/O:5 (27), 2/O:9, 3/O:3, and 4/O:3 (Hannu et al., 2003). Y. enterocolitica strains have been isolated from a variety of domestic animals, especially from the tonsils and tongues of pigs (Fredriksson-Ahomaa and Korkeala, 2003; Fredriksson-Ahomaa et al., 2001, 2003, 2002). Porcine products are considered to be the primary source of this pathogen in humans (Bottone, 1999). In recent years, many methods have been developed for the detection of the pathogenic Y. enterocolitica strain. PCR and real-time PCR methods were developed as efficient tools for identifying strains of Y. enterocolitica and Yersinia spp. (Kechagia et al., 2007; Kot et al., 2007). Pulsed-field gel electrophoresis was used to distinguish the pathogenic Yersinia isolates from the nonpathogenic ones (Thisted Lambertz and Danielsson-Tham, 2005). A novel density gradient centrifugation method named flotation was used to prepare the DNA samples prior to PCR (Wolffs et al., 2004). Methods involve in a bacterial culture enrichment as well as PCR for the detection of Y. enterocolitica have been evaluated (Lambertz et al., 2007).
⁎ Corresponding author. E-mail address: [email protected] (H. Gao). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.02.001
Loop-mediated isothermal amplification (LAMP) is an alternative method that is based on DNA amplification (Notomi et al., 2000). The LAMP method amplifies DNA with a high degree of specificity, efficiency, and rapidity under isothermal conditions. LAMP assays have been developed for the detection of Escherichia coli (Hara-Kudo et al., 2007), Salmonella spp. (Hara-Kudo et al., 2005) and other organisms. In this study, the efficiency of the LAMP method was tested by using cultured Y. enterocolitica strains, including the bioserotypes 1B/O:8, 2/O:5,27, 2/O:9, 3/O:3, and 4/O:3. Furthermore, the specificity and sensitivity of the LAMP method were evaluated by comparing the performances of the LAMP method with the method combining PCR and bacterial culture enrichment. The samples used for this comparison were 21 pig tonsil samples and 73 pork meat samples obtained from 94 slaughtered pigs belonging to 4 different herds. 2. Materials and methods 2.1. Bacterial strains and DNA preparation The Y. enterocolitica strains used in this study are listed in Table 1. These strains were used to design the specificity of the LAMP primers before applying the raw meat test. A known quantity of the Y. enterocolitica (IQCC10903) strain was suspended in 4 mL of peptone water and mixed carefully before 10-fold serial dilution. Each dilution was separated into 2 aliquots immediately after being mixed. The number of Y. enterocolitica cells in 1 aliquot of the highest and the lowest bacterial dilution were determined by viable counts on nutrient agar plates, which were incubated overnight at 30 °C.
H. Gao et al. / Journal of Microbiological Methods 77 (2009) 198–201
199
Table 1 List of bacterial strains and their sources that were used in the Y. enterocolitica LAMP experiment. Species
Strains number
Bioserotype and source
Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. pseudotuberculosis Y. frederiksenii Escherichia coli Salmonella enteritidis Salmonella typhi Shigella flexneri Shigella sonnei Shigella bogdii Clostridium perfrigens Listeria monocytogenes Vibrio parahaemolyticus Listeria ivanovii Listeria grayi Listeria seeligeri Listeria innocua Listeria welshimeri Staphylococcus aureus Raoultella planticola Klebsiella oxytoca Citrobacter freundii Citrobacter braakii
IQCC10904 IQCC10901 IQCC10902 IQCC10903 IQCC10907 CMCC52211 CMCC 52212 CMCC 52203 CMCC53504 ATCC 33641 IQCC10102 IQCC10534 IQCC10593 IQCC11304 IQCC11305 IQCC11306 IQCC23901 IQCC22226 IQCC12310 IQCC22227 IQCC22228 IQCC22229 IQCC22260 IQCC22261 IQCC22045 20080913-1 20080913-2 20080913-3 20080913-4
2/O:9, donated by aCAIQ 4/O:3, donated by aCAIQ 2/O:5.27, donated by aCAIQ 2/O:8, donated by aCAIQ 1/O:8, donated by aCAIQ 1/O:8, purchased from bCMCC 2/O:9, purchased from bCMCC 4/O:3, purchased from bCMCC purchased from bCMCC purchased from dATCC donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ c SDCIQ, isolated from pig tonsil, identified c SDCIQ, isolated from pig tonsil, identified c SDCIQ, isolated from pig tonsil, identified c SDCIQ, isolated from pig tonsil, identified
a b c d
by VITEK 2 by VITEK 2 by VITEK 2 by VITEK 2
Compact Compact Compact Compact
(bioMerieux, (bioMerieux, (bioMerieux, (bioMerieux,
France), 2008 France), 2008 France), 2008 France), 2008
CAIQ, Chinese Academy of Inspection and Quarantine, Beijing, China. CMCC, China Center of Medical Microbiology Culture Collective, Beijing, China. SDCIQ, Shandong Entry–Exit Inspection and Quarantine Bureau, Qingdao, China. ATCC, American Type Culture Collection 10801 University Boulevard Manassas (VA), USA.
2.2. Primers and LAMP condition The sequences of the LAMP primers that were used in the current study were designed by the PrimerExplorer V4 software program (http://primerexplorer.jp/elamp4.0.0/index.html) according to the sequences present in the gyrB gene (GenBank access number, DQ386883.1, EF175580.1, AB084027.1).The primers used in LAMP detection included: Y-BIP primer: 5-CCGGTTTGATCGGTTTCGCCCACTTA CAAGATGGGTGTGCC-3), Y-FIP: 5-GTGCGTTTCTGGCCGAGCTTGCAGA CGTTTTGCCAGGATT-3), Y-B3: 5-CGCCGTGAAGGTAAAGTTCA-3 and Y-F3: 5-CAGAGTT-CAGGAACGACAGC-3. The LAMP assay was performed in a total of 25 µL of the reaction mixture containing 30 pmol of the primers Y-FIP and Y-BIP, 4 pmol of the primers Y-F3 and Y-B3, 2.5 µL of a 10× reaction mixture (New England
Biolabs), 12 mmol/L MgSO4, 1.0 M betaine, 1.0 mmol/L of deoxynucleotide triphosphates (dGTP, dATP, dCTP, and dTTP), 2 µL of the template DNA, and 8 U of Bst DNA polymerase (New England Biolabs). The reaction mixture was incubated at 65 °C for 60 min and then at 80 °C for 4 min in order to facilitate the termination of the reaction. Each product (4 µL) was analyzed by electrophoresis on a 2.0% agarose gel that was stained with GelRed (Biotium, USA). The LAMP products were also detected visually on the basis of a color that indicated a color change (from orange to yellow) after the addition of PicoGreen (Invitrogen, Carlsbad, USA) dye (2 µL) to the tubes containing the LAMP products (Tomlinson et al., 2007). PCR was carried out in a final volume of 25 µL in 0.2 mL tubes that contained 10 pmol of the primers Y-F3 and Y-B3, a 10× reaction buffer B (Promega, Wisconsin USA), 2 mmol/L of MgCl2, 200 nmol/L
Fig. 1. The LAMP specific amplification for gyrB gene of Y. enterorcolitica. Lanes 1 to 8: 8 brains of Y. enterocolitica as Table 1; Lanes 9: Y. pseudotuberculosis; Lane 10: Y. frederiksenii; Lanes 11: Escherichia coli;Lane 12: Salmonella enteritidis; Lanes 13: Salmonella typhi; Lane 14: Shigella flexneri; Lanes 15: Shigella sonne; Lane 16:Shigella bogdii; Lanes 17: Clostridium perfrigens; Lane 18: Listeria monocytogenes; Lanes 19: Vibrio parahaemolyticus; Lane 20:Listeria ivanovi;i Lanes 21: Listeria grayi; Lane 22: Listeria seeligeri; Lanes 23: Listeria innocua; Lane 24: no template control. M: DL 2000 (Takara, China) was used as molecular weight marker; fragments from top to bottom: 2000, 1000, 750, 500, 250, and 100 bp.
200
H. Gao et al. / Journal of Microbiological Methods 77 (2009) 198–201
sterile distilled water and DNA was subsequently extracted using the DNA extraction kit (Tiangen, China). Simultaneously, the other sample set was cultured by one of the standard culture methods, ISO 10273 (Lambertz et al., 2007). A nonselective peptone sorbit bile enrichment broth at 25 °C for 48 h using 1/10 dilution samples was followed by a transfer of 0.5 mL of the culture to 4.5 mL of 0.5% KOH in order to inhibit the growth of background flora. The cultures were then incubated on selective cefsulodin–irgasan–novobiocin (CIN) agar suspected colonies were plated on CIN agar and were further identified with the API 20E system (bioMerieux). 3. Results 3.1. Evaluation of the LAMP method using artificially inoculated samples
Fig. 2. (a) The LAMP amplification sensitivity for gyrB gene was estimated using Y. enterorcolitica (IQCC10903). Lanes 1 and 2: 6.5 × 107 CPU Y. enterorcolitica; Lanes 3 and 4: about 6.5× 106 CPU Y. enterorcolitica; Lanes 5 and 6: about 6.5× 105 CPU Y. enterorcolitica; Lanes 7 and 8: about 6.5 × 104 CPU Y. enterorcolitica; Lanes 9 and 10: about 6.5× 103 CPU Y. enterorcolitica; Lanes 11 and 12: about 65 CPU Y. enterorcolitica; Lanes 13 and 14: 4 CPU Y. enterorcolitica; Lanes 15 and 16: no template control. M: DL 2000 (Takara, China) was used as molecular weight marker; fragments from top to bottom: 2000, 1000, 750, 500, 250, and 100 bp. (b) The PCR amplification sensitivity for gyrB gene was estimated using Y. enterorcolitica (IQCC10903). Lanes 1: 6.5 × 107 CPU Y. enterorcolitica; Lanes 2: about 6.5 × 106 CPU Y. enterorcolitica; Lanes 3: about 6.5 × 105 CPU Y. enterorcolitica; Lanes 4: about 6.5 × 104 CPU Y. enterorcolitica; Lanes 5: about 6.5 × 103 CPU Y. enterorcolitica; Lanes 6: about 65 CPU Y. enterorcolitica; Lanes 7: 4 CPU Y. enterorcolitica; Lanes 8: no template control. M: DL 2000.
deoxynucleotide triphosphates, 1.5 U of Taq DNA polymerase, and 1.0 µL of the DNA template. After 30 s of initial denaturation at 94 °C, the PCR conditions were optimized as follows: 35 cycles of amplification (at 94 °C for 30 s, at 56 °C for 30 s, and at 72 °C for 30 s), and a final extension at 72 °C for 4 min. Each PCR product (6 µL) was subsequently analyzed by electrophoresis on 2.0% agarose gels that were stained with GelRed (Biotium, USA).
2.3. Pathogenic Y. enterocolitica in pig samples The 21 pig tonsil samples and 73 pork meat samples were obtained as unfrozen meat from 94 slaughtered pigs belonging to 4 different local herds. All the samples were separated into 2 sets; the samples of 1 set were minced, placed in 10 mL of peptone sorbit bile enrichment broth, and then incubated at 25 °C for 48 h (Lambertz et al., 2007). The 48-h-old culture (10 mL) was then centrifuged at 12000 g for 4 min. The pellet obtained on centrifugation was suspended in 100 µL of
The selectivity test for the LAMP method was performed using 8 Y. enterocolitica strains (5 bioserotypes), 2 other Yersinia strains, and 19 non-Yersinia bacterial strains. All 8 Y. enterocolitica strains were positively detected by both the PCR and LAMP methods (Fig. 1); No other bacterial strain was detected or amplified by LAMP. When 2 µL of PicoGreen was added to the LAMP products, the resulting color change observed was orange to yellow (Fig. 3) in 1–3 min. The sensitivity of the LAMP method was determined using the DNA of the serially diluted overnight culture of Y. enterocolitica. The LAMP method could detect Y. enterocolitica at a dilution of 65 CFU/mL. The detection limit of both the LAMP (Fig. 2(a)) and PCR (Fig. 2(b)) assays for Y. enterocolitica was approximately 31.6 fg DNA/reaction. 3.2. Evaluation of the LAMP method on raw pork meat Of the total of 94 pig samples that were analyzed, 4 (24%) tonsil samples and none of the meat samples tested positively for Y. enterocolitica by LAMP and PCR. However, none of the Y. enterocolitica strains was isolated in the bacterial culture enrichment method. The color of the 4 LAMP positive products was observed to change from orange to yellow after the addition of 2 µL of PicoGreen dye to the LAMP reaction tubes in 1 to 3 min (Fig. 3). 4. Discussion Methods that involve the detection of specific DNA sequences have been applied previously to detect the pathogenic Y. enterocolitica and Y. pseudotuberculosis strains (Cocolin and Comi, 2005; Kot et al., 2007; Myers et al., 2006; Wolffs et al., 2004). Further, multiplex PCR methods that use the ail, yadA, ystB, and inv genes were developed in order to discriminate the different bioserotypes of Y. enterocolitica and Y. pseudotuberculosis (Kot et al., 2007). However, the application of the LAMP method has not been reported yet. The LAMP primers that we used to detect Y. enterocolitica were based on gyrB sequences which present in all common Y. enterocolitica bioserotypes known to cause infections in humans. Moreover, the
Fig. 3. Detection of Y. enterorcolitica from raw pork by LAMP by adding 2 µL PicoGreen dsDNA reagent. The presence of a large amount of LAMP product in positive reaction mixtures causes a color change from orange to yellow. Tube 1 and 2: the raw pork samples for Y. enterorcolitica negative; Tube 3–6: the raw pork samples for Y. enterorcolitica passive.
H. Gao et al. / Journal of Microbiological Methods 77 (2009) 198–201
primers have also been shown to be specific to Y. enterocolitica strains, because no LAMP products were obtained when detecting Y. enterocolitica from a mixed culture containing other bacterial strains, such as Y. pseudotuberculosis, Y. frederiksenii, Salmonella enteritidis, Shigella flexneri, Escherichia coli, and Clostridium perfringens strains (Fig. 1). This DNA-based assay enabled detection of Y. enterocolitica in a significantly shorter time period. Of the 94 raw pork samples examined, 4 tested positive by the LAMP method after overnight enrichment; no Y. enterocolitica bacteria were grown from those samples with the standard culture method (ISO 10273). In the study that involved a combination of the bacterial culture enrichment and PCR methods for the detection of Y. enterocolitica (Lambertz et al., 2007), of the 97 pork sausage samples analyzed, 11% tested positive by PCR. The enrichment step increased the possibility of detecting live bacteria (Lambertz et al., 2007). A drawback of DNA amplification-based techniques is the possible detection of dead or damaged cells along with the live cells. This can be rectified by the enrichment step or by the addition of DNase before the cell lysis step (Nogva et al., 2000). In summary, the LAMP method is a viable alternative for the rapid detection of Y. enterocolitica in food. The LAMP method could be further exploited in field tests with inexpensive equipment because the LAMP assay can be carried out under isothermal conditions at 65 °C. Acknowledgments This work was supported by Major State Basic Research Development Program of China 2006BAD05A06-01 and research project of the General Administration of Quality Supervision, Inspection and Quarantine of P.R. China: 2007IK167. References Bottone, E.J., 1999. Yersinia enterocolitica: overview and epidemiologic correlates. Microbes Infect. 1, 323–333. Cocolin, L., Comi, G., 2005. Use of a culture-independent molecular method to study the ecology of Yersinia spp. in food. Int. J. Food Microbiol. 105, 71–82.
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Fredriksson-Ahomaa, M., Korkeala, H., 2003. Low occurrence of pathogenic Yersinia enterocolitica in clinical, food, and environmental samples: a methodological problem. Clin. Microbiol. Rev. 16, 220–229. Fredriksson-Ahomaa, M., Korte, T., Korkeala, H., 2001. Transmission of Yersinia enterocolitica 4/O:3 to pets via contaminated pork. Lett. Appl. Microbiol. 32, 375–378. Fredriksson-Ahomaa, M., Niskanen, T., Bucher, M., Korte, T., Stolle, A., Korkeala, H., 2003. Different Yersinia enterocolitica 4:O3 genotypes found in pig tonsils in Southern Germany and Finland. Syst. Appl. Microbiol. 26, 132–137. Fredriksson-Ahomaa, M., Niskanen, T., Neubauer, H., Laukkanen, R., Korkeala, H., 2002. Characterisation of sucrose-negative Yersinia enterocolitica 4/O:3 isolates recovered from pig tonsils. Int. J. Food Microbiol. 75, 19–25. Hannu, T., Mattila, L., Nuorti, J.P., Ruutu, P., Mikkola, J., Siitonen, A., Leirisalo-Repo, M., 2003. Reactive arthritis after an outbreak of Yersinia pseudotuberculosis serotype O:3 infection. Ann. Rheum. Dis. 62, 866–869. Hara-Kudo, Y., Nemoto, J., Ohtsuka, K., Segawa, Y., Takatori, K., Kojima, T., Ikedo, M., 2007. Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loopmediated isothermal amplification. J. Med. Microbiol. 56, 398–406. Hara-Kudo, Y., Yoshino, M., Kojima, T., Ikedo, M., 2005. Loop-mediated isothermal amplification for the rapid detection of Salmonella. FEMS Microbiol. Lett. 253,155–161. Kechagia, N., Nicolaou, C., Ioannidou, V., Kourti, E., Ioannidis, A., Legakis, N.J., Chatzipanagiotou, S., 2007. Detection of chromosomal and plasmid-encoded virulence determinants in Yersinia enterocolitica and other Yersinia spp. isolated from food animals in Greece. Int. J. Food Microbiol. 118, 326–331. Kot, B., Trafny, E.A., Jakubczak, A., 2007. Application of multiplex PCR for monitoring colonization of pig tonsils by Yersinia enterocolitica, including biotype 1A, and Yersinia pseudotuberculosis. J. Food Prot. 70, 1110–1115. Lambertz, S.T., Granath, K., Fredriksson-Ahomaa, M., Johansson, K.E., Danielsson-Tham, M.L., 2007. Evaluation of a combined culture and PCR method (NMKL-163A) for detection of presumptive pathogenic Yersinia enterocolitica in pork products. J. Food Prot. 70, 335–340. Myers, K.M., Gaba, J., Al-Khaldi, S.F., 2006. Molecular identification of Yersinia enterocolitica isolated from pasteurized whole milk using DNA microarray chip hybridization. Mol. Cell. Probes 20, 71–80. Nogva, H.K., Bergh, A., Holck, A., Rudi, K., 2000. Application of the 5′-nuclease PCR assay in evaluation and development of methods for quantitative detection of Campylobacter jejuni. Appl. Environ. Microbiol. 66, 4029–4036. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T., 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, E63. Thisted Lambertz, S., Danielsson-Tham, M.L., 2005. Identification and characterization of pathogenic Yersinia enterocolitica isolates by PCR and pulsed-field gel electrophoresis. Appl. Environ. Microbiol. 71, 3674–3681. Tomlinson, J.A., Barker, I., Boonham, N., 2007. Faster, simpler, more-specific methods for improved molecular detection of phytophthora ramorum in the field. Appl. Environ. Mocrobiol. 73, 4040–4047. Wolffs, P., Knutsson, R., Norling, B., Radstrom, P., 2004. Rapid quantification of Yersinia enterocolitica in pork samples by a novel sample preparation method, flotation, prior to real-time PCR. J. Clin. Microbiol. 42, 1042–1047.
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Application of loop-mediated isothermal amplification for detection of Yersinia enterocolitica in pork meat Hongwei Gao a,⁎, Zhiwen Lei a, Juntao Jia a, Shufeng Wang a, Ying Chen b, Min Sun a, Chengzhu Liang a a b
Shandong Entry–Exit Inspection and Quarantine Bureau of the People's Republic of China, Qingdao, China Chinese Academy of Inspection and Quarantine, Institute of Food Safety, Beijing, China
a r t i c l e
i n f o
Article history: Received 13 October 2008 Received in revised form 3 February 2009 Accepted 3 February 2009 Available online 12 February 2009 Keywords: Loop-mediated isothermal amplification Detection Yersinia enterocolitica
a b s t r a c t A loop-mediated isothermal amplification (LAMP) method was developed for the detection of Yersinia enterocolitica isolates in both pure bacterial cultures and pork meat. The LAMP primers, which corresponded to the gyrB gene, accurately identified 4 different bioserotypes of Y. enterocolitica. These primers failed to detect Y. pseudotuberculosis, Y. frederiksenii, and 17 non-Yersinia strains. The sensitivity of the LAMP assay for the detection of Y. enterocolitica in pure culture was 65 CFU/mL (31.6 fg of genomic DNA). The LAMP assay was conducted for the detection of Y. enterocolitica strains in 21 pig tonsil samples and 73 pork meat samples obtained from 94 slaughtered pigs belonging to 4 different herds. Y. enterocolitica was found to be present in 4 tonsil samples and none in meat samples. This is the first report in which the LAMP assay was employed for the detection of Y. enterocolitica in food samples. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Yersinia enterocolitica is a common enteric pathogen in humans. This pathogen has a strong propensity to cause acute gastroenteritis, enterocolitis, and mesenteric adenitis, as well as a variety of extraintestinal disorders (Bottone, 1999). This organism has been classified into approximately 60 serotypes. However, most strains that are associated with human yersiniosis belong to the bioserotypes 1B/O:8, 2/O:5 (27), 2/O:9, 3/O:3, and 4/O:3 (Hannu et al., 2003). Y. enterocolitica strains have been isolated from a variety of domestic animals, especially from the tonsils and tongues of pigs (Fredriksson-Ahomaa and Korkeala, 2003; Fredriksson-Ahomaa et al., 2001, 2003, 2002). Porcine products are considered to be the primary source of this pathogen in humans (Bottone, 1999). In recent years, many methods have been developed for the detection of the pathogenic Y. enterocolitica strain. PCR and real-time PCR methods were developed as efficient tools for identifying strains of Y. enterocolitica and Yersinia spp. (Kechagia et al., 2007; Kot et al., 2007). Pulsed-field gel electrophoresis was used to distinguish the pathogenic Yersinia isolates from the nonpathogenic ones (Thisted Lambertz and Danielsson-Tham, 2005). A novel density gradient centrifugation method named flotation was used to prepare the DNA samples prior to PCR (Wolffs et al., 2004). Methods involve in a bacterial culture enrichment as well as PCR for the detection of Y. enterocolitica have been evaluated (Lambertz et al., 2007).
⁎ Corresponding author. E-mail address: [email protected] (H. Gao). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.02.001
Loop-mediated isothermal amplification (LAMP) is an alternative method that is based on DNA amplification (Notomi et al., 2000). The LAMP method amplifies DNA with a high degree of specificity, efficiency, and rapidity under isothermal conditions. LAMP assays have been developed for the detection of Escherichia coli (Hara-Kudo et al., 2007), Salmonella spp. (Hara-Kudo et al., 2005) and other organisms. In this study, the efficiency of the LAMP method was tested by using cultured Y. enterocolitica strains, including the bioserotypes 1B/O:8, 2/O:5,27, 2/O:9, 3/O:3, and 4/O:3. Furthermore, the specificity and sensitivity of the LAMP method were evaluated by comparing the performances of the LAMP method with the method combining PCR and bacterial culture enrichment. The samples used for this comparison were 21 pig tonsil samples and 73 pork meat samples obtained from 94 slaughtered pigs belonging to 4 different herds. 2. Materials and methods 2.1. Bacterial strains and DNA preparation The Y. enterocolitica strains used in this study are listed in Table 1. These strains were used to design the specificity of the LAMP primers before applying the raw meat test. A known quantity of the Y. enterocolitica (IQCC10903) strain was suspended in 4 mL of peptone water and mixed carefully before 10-fold serial dilution. Each dilution was separated into 2 aliquots immediately after being mixed. The number of Y. enterocolitica cells in 1 aliquot of the highest and the lowest bacterial dilution were determined by viable counts on nutrient agar plates, which were incubated overnight at 30 °C.
H. Gao et al. / Journal of Microbiological Methods 77 (2009) 198–201
199
Table 1 List of bacterial strains and their sources that were used in the Y. enterocolitica LAMP experiment. Species
Strains number
Bioserotype and source
Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. enterocolitica Y. pseudotuberculosis Y. frederiksenii Escherichia coli Salmonella enteritidis Salmonella typhi Shigella flexneri Shigella sonnei Shigella bogdii Clostridium perfrigens Listeria monocytogenes Vibrio parahaemolyticus Listeria ivanovii Listeria grayi Listeria seeligeri Listeria innocua Listeria welshimeri Staphylococcus aureus Raoultella planticola Klebsiella oxytoca Citrobacter freundii Citrobacter braakii
IQCC10904 IQCC10901 IQCC10902 IQCC10903 IQCC10907 CMCC52211 CMCC 52212 CMCC 52203 CMCC53504 ATCC 33641 IQCC10102 IQCC10534 IQCC10593 IQCC11304 IQCC11305 IQCC11306 IQCC23901 IQCC22226 IQCC12310 IQCC22227 IQCC22228 IQCC22229 IQCC22260 IQCC22261 IQCC22045 20080913-1 20080913-2 20080913-3 20080913-4
2/O:9, donated by aCAIQ 4/O:3, donated by aCAIQ 2/O:5.27, donated by aCAIQ 2/O:8, donated by aCAIQ 1/O:8, donated by aCAIQ 1/O:8, purchased from bCMCC 2/O:9, purchased from bCMCC 4/O:3, purchased from bCMCC purchased from bCMCC purchased from dATCC donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ donated by aCAIQ c SDCIQ, isolated from pig tonsil, identified c SDCIQ, isolated from pig tonsil, identified c SDCIQ, isolated from pig tonsil, identified c SDCIQ, isolated from pig tonsil, identified
a b c d
by VITEK 2 by VITEK 2 by VITEK 2 by VITEK 2
Compact Compact Compact Compact
(bioMerieux, (bioMerieux, (bioMerieux, (bioMerieux,
France), 2008 France), 2008 France), 2008 France), 2008
CAIQ, Chinese Academy of Inspection and Quarantine, Beijing, China. CMCC, China Center of Medical Microbiology Culture Collective, Beijing, China. SDCIQ, Shandong Entry–Exit Inspection and Quarantine Bureau, Qingdao, China. ATCC, American Type Culture Collection 10801 University Boulevard Manassas (VA), USA.
2.2. Primers and LAMP condition The sequences of the LAMP primers that were used in the current study were designed by the PrimerExplorer V4 software program (http://primerexplorer.jp/elamp4.0.0/index.html) according to the sequences present in the gyrB gene (GenBank access number, DQ386883.1, EF175580.1, AB084027.1).The primers used in LAMP detection included: Y-BIP primer: 5-CCGGTTTGATCGGTTTCGCCCACTTA CAAGATGGGTGTGCC-3), Y-FIP: 5-GTGCGTTTCTGGCCGAGCTTGCAGA CGTTTTGCCAGGATT-3), Y-B3: 5-CGCCGTGAAGGTAAAGTTCA-3 and Y-F3: 5-CAGAGTT-CAGGAACGACAGC-3. The LAMP assay was performed in a total of 25 µL of the reaction mixture containing 30 pmol of the primers Y-FIP and Y-BIP, 4 pmol of the primers Y-F3 and Y-B3, 2.5 µL of a 10× reaction mixture (New England
Biolabs), 12 mmol/L MgSO4, 1.0 M betaine, 1.0 mmol/L of deoxynucleotide triphosphates (dGTP, dATP, dCTP, and dTTP), 2 µL of the template DNA, and 8 U of Bst DNA polymerase (New England Biolabs). The reaction mixture was incubated at 65 °C for 60 min and then at 80 °C for 4 min in order to facilitate the termination of the reaction. Each product (4 µL) was analyzed by electrophoresis on a 2.0% agarose gel that was stained with GelRed (Biotium, USA). The LAMP products were also detected visually on the basis of a color that indicated a color change (from orange to yellow) after the addition of PicoGreen (Invitrogen, Carlsbad, USA) dye (2 µL) to the tubes containing the LAMP products (Tomlinson et al., 2007). PCR was carried out in a final volume of 25 µL in 0.2 mL tubes that contained 10 pmol of the primers Y-F3 and Y-B3, a 10× reaction buffer B (Promega, Wisconsin USA), 2 mmol/L of MgCl2, 200 nmol/L
Fig. 1. The LAMP specific amplification for gyrB gene of Y. enterorcolitica. Lanes 1 to 8: 8 brains of Y. enterocolitica as Table 1; Lanes 9: Y. pseudotuberculosis; Lane 10: Y. frederiksenii; Lanes 11: Escherichia coli;Lane 12: Salmonella enteritidis; Lanes 13: Salmonella typhi; Lane 14: Shigella flexneri; Lanes 15: Shigella sonne; Lane 16:Shigella bogdii; Lanes 17: Clostridium perfrigens; Lane 18: Listeria monocytogenes; Lanes 19: Vibrio parahaemolyticus; Lane 20:Listeria ivanovi;i Lanes 21: Listeria grayi; Lane 22: Listeria seeligeri; Lanes 23: Listeria innocua; Lane 24: no template control. M: DL 2000 (Takara, China) was used as molecular weight marker; fragments from top to bottom: 2000, 1000, 750, 500, 250, and 100 bp.
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sterile distilled water and DNA was subsequently extracted using the DNA extraction kit (Tiangen, China). Simultaneously, the other sample set was cultured by one of the standard culture methods, ISO 10273 (Lambertz et al., 2007). A nonselective peptone sorbit bile enrichment broth at 25 °C for 48 h using 1/10 dilution samples was followed by a transfer of 0.5 mL of the culture to 4.5 mL of 0.5% KOH in order to inhibit the growth of background flora. The cultures were then incubated on selective cefsulodin–irgasan–novobiocin (CIN) agar suspected colonies were plated on CIN agar and were further identified with the API 20E system (bioMerieux). 3. Results 3.1. Evaluation of the LAMP method using artificially inoculated samples
Fig. 2. (a) The LAMP amplification sensitivity for gyrB gene was estimated using Y. enterorcolitica (IQCC10903). Lanes 1 and 2: 6.5 × 107 CPU Y. enterorcolitica; Lanes 3 and 4: about 6.5× 106 CPU Y. enterorcolitica; Lanes 5 and 6: about 6.5× 105 CPU Y. enterorcolitica; Lanes 7 and 8: about 6.5 × 104 CPU Y. enterorcolitica; Lanes 9 and 10: about 6.5× 103 CPU Y. enterorcolitica; Lanes 11 and 12: about 65 CPU Y. enterorcolitica; Lanes 13 and 14: 4 CPU Y. enterorcolitica; Lanes 15 and 16: no template control. M: DL 2000 (Takara, China) was used as molecular weight marker; fragments from top to bottom: 2000, 1000, 750, 500, 250, and 100 bp. (b) The PCR amplification sensitivity for gyrB gene was estimated using Y. enterorcolitica (IQCC10903). Lanes 1: 6.5 × 107 CPU Y. enterorcolitica; Lanes 2: about 6.5 × 106 CPU Y. enterorcolitica; Lanes 3: about 6.5 × 105 CPU Y. enterorcolitica; Lanes 4: about 6.5 × 104 CPU Y. enterorcolitica; Lanes 5: about 6.5 × 103 CPU Y. enterorcolitica; Lanes 6: about 65 CPU Y. enterorcolitica; Lanes 7: 4 CPU Y. enterorcolitica; Lanes 8: no template control. M: DL 2000.
deoxynucleotide triphosphates, 1.5 U of Taq DNA polymerase, and 1.0 µL of the DNA template. After 30 s of initial denaturation at 94 °C, the PCR conditions were optimized as follows: 35 cycles of amplification (at 94 °C for 30 s, at 56 °C for 30 s, and at 72 °C for 30 s), and a final extension at 72 °C for 4 min. Each PCR product (6 µL) was subsequently analyzed by electrophoresis on 2.0% agarose gels that were stained with GelRed (Biotium, USA).
2.3. Pathogenic Y. enterocolitica in pig samples The 21 pig tonsil samples and 73 pork meat samples were obtained as unfrozen meat from 94 slaughtered pigs belonging to 4 different local herds. All the samples were separated into 2 sets; the samples of 1 set were minced, placed in 10 mL of peptone sorbit bile enrichment broth, and then incubated at 25 °C for 48 h (Lambertz et al., 2007). The 48-h-old culture (10 mL) was then centrifuged at 12000 g for 4 min. The pellet obtained on centrifugation was suspended in 100 µL of
The selectivity test for the LAMP method was performed using 8 Y. enterocolitica strains (5 bioserotypes), 2 other Yersinia strains, and 19 non-Yersinia bacterial strains. All 8 Y. enterocolitica strains were positively detected by both the PCR and LAMP methods (Fig. 1); No other bacterial strain was detected or amplified by LAMP. When 2 µL of PicoGreen was added to the LAMP products, the resulting color change observed was orange to yellow (Fig. 3) in 1–3 min. The sensitivity of the LAMP method was determined using the DNA of the serially diluted overnight culture of Y. enterocolitica. The LAMP method could detect Y. enterocolitica at a dilution of 65 CFU/mL. The detection limit of both the LAMP (Fig. 2(a)) and PCR (Fig. 2(b)) assays for Y. enterocolitica was approximately 31.6 fg DNA/reaction. 3.2. Evaluation of the LAMP method on raw pork meat Of the total of 94 pig samples that were analyzed, 4 (24%) tonsil samples and none of the meat samples tested positively for Y. enterocolitica by LAMP and PCR. However, none of the Y. enterocolitica strains was isolated in the bacterial culture enrichment method. The color of the 4 LAMP positive products was observed to change from orange to yellow after the addition of 2 µL of PicoGreen dye to the LAMP reaction tubes in 1 to 3 min (Fig. 3). 4. Discussion Methods that involve the detection of specific DNA sequences have been applied previously to detect the pathogenic Y. enterocolitica and Y. pseudotuberculosis strains (Cocolin and Comi, 2005; Kot et al., 2007; Myers et al., 2006; Wolffs et al., 2004). Further, multiplex PCR methods that use the ail, yadA, ystB, and inv genes were developed in order to discriminate the different bioserotypes of Y. enterocolitica and Y. pseudotuberculosis (Kot et al., 2007). However, the application of the LAMP method has not been reported yet. The LAMP primers that we used to detect Y. enterocolitica were based on gyrB sequences which present in all common Y. enterocolitica bioserotypes known to cause infections in humans. Moreover, the
Fig. 3. Detection of Y. enterorcolitica from raw pork by LAMP by adding 2 µL PicoGreen dsDNA reagent. The presence of a large amount of LAMP product in positive reaction mixtures causes a color change from orange to yellow. Tube 1 and 2: the raw pork samples for Y. enterorcolitica negative; Tube 3–6: the raw pork samples for Y. enterorcolitica passive.
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primers have also been shown to be specific to Y. enterocolitica strains, because no LAMP products were obtained when detecting Y. enterocolitica from a mixed culture containing other bacterial strains, such as Y. pseudotuberculosis, Y. frederiksenii, Salmonella enteritidis, Shigella flexneri, Escherichia coli, and Clostridium perfringens strains (Fig. 1). This DNA-based assay enabled detection of Y. enterocolitica in a significantly shorter time period. Of the 94 raw pork samples examined, 4 tested positive by the LAMP method after overnight enrichment; no Y. enterocolitica bacteria were grown from those samples with the standard culture method (ISO 10273). In the study that involved a combination of the bacterial culture enrichment and PCR methods for the detection of Y. enterocolitica (Lambertz et al., 2007), of the 97 pork sausage samples analyzed, 11% tested positive by PCR. The enrichment step increased the possibility of detecting live bacteria (Lambertz et al., 2007). A drawback of DNA amplification-based techniques is the possible detection of dead or damaged cells along with the live cells. This can be rectified by the enrichment step or by the addition of DNase before the cell lysis step (Nogva et al., 2000). In summary, the LAMP method is a viable alternative for the rapid detection of Y. enterocolitica in food. The LAMP method could be further exploited in field tests with inexpensive equipment because the LAMP assay can be carried out under isothermal conditions at 65 °C. Acknowledgments This work was supported by Major State Basic Research Development Program of China 2006BAD05A06-01 and research project of the General Administration of Quality Supervision, Inspection and Quarantine of P.R. China: 2007IK167. References Bottone, E.J., 1999. Yersinia enterocolitica: overview and epidemiologic correlates. Microbes Infect. 1, 323–333. Cocolin, L., Comi, G., 2005. Use of a culture-independent molecular method to study the ecology of Yersinia spp. in food. Int. J. Food Microbiol. 105, 71–82.
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