Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Development of a loop-mediated isothermal amplification assay for rapid and simple detection of Erysipelothrix rhusiopathiae Y. Yamazaki1, E. Oba2, N. Kashiwagi2, K. Sugita3, K. Shiiba3, Y. Baba1, Y. Shimoji4 and W. Yamazaki5,6 1 2 3 4 5 6

Kobayashi Meat Inspection Office, Miyazaki, Japan Miyakonojyo Meat Inspection Office, Miyazaki, Japan Tsuno Meat Inspection Office, Miyazaki, Japan National Institute of Animal Health, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan Center for Animal Disease Control, University of Miyazaki, Miyazaki, Japan

Significance and Impact of the Study: This is the first report of a loop-mediated isothermal amplification (LAMP) assay for simple and cost-effective detection of E. rhusiopathiae from swine samples. The LAMP assay provided more rapid detection of the bacterium than conventional PCR and biochemicalbased assays, and it may potentially facilitate surveillance and early diagnosis of swine erysipelas in the field.

Keywords Erysipelothrix rhusiopathiae, loop-mediated isothermal amplification, rapid detection, swine erysipelas. Correspondence Wataru Yamazaki, Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen Kibanadai-nishi, Miyazaki, Miyazaki, 889-2192 Japan. E-mail: [email protected] 2013/1305: received 28 June 2013, revised 17 November 2013 and accepted 17 November 2013 doi:10.1111/lam.12198

Abstract Erysipelothrix rhusiopathiae is a causative agent of swine erysipelas. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of E. rhusiopathiae. The LAMP assay correctly detected 39 E. rhusiopathiae strains. No LAMP products were detected from 14 non-rhusiopathiae Erysipelothrix and 16 non-Erysipelothrix strains, including E. tonsillarum serovar 10 strains, which are difficult to be discriminated from E. rhusiopathiae strains. These results were consistent with those obtained by a conventional E. rhusiopathiae-specific PCR assay. Starting with DNA extraction from a single colony, the gel-based PCR assay took 4 h to provide a result, but the LAMP assay was faster, requiring only 37–80 min. The conventional culture test required more than 3–4 days to isolate and identify E. rhusiopathiae in the enrichment cultures. In contrast, the LAMP assay required less than 22 h from the beginning of the enrichment culture to final determination. These results suggest that the LAMP assay is useful as an adjunct to facilitate early diagnosis of swine erysipelas.

Introduction Erysipelothrix rhusiopathiae, which is widely distributed in nature, causes erysipelas in swine and a variety of diseases in other animals and birds, as well as erysipeloid, a skin disease of humans. Swine erysipelas, a disease causing enormous economic losses in the swine industry throughout the world, can occur as an acute septicaemia or chronic disease with development of arthritic lesions, polyarthritis, lymphadenitis and endocarditis (Makino et al. 362

1994; Shimoji et al. 1998, 2000; Wood 1999; Pal et al. 2010; Wang et al. 2010). The genus Erysipelothrix has long been thought to be represented by the single species E. rhusiopathiae, which was classified into 25 serovars on the basis of peptidoglycan antigens of the cell wall (Takahashi et al. 1992). To date, DNA–DNA hybridization analysis clarified the genus comprising at least the two species, E. rhusiopathiae and Erysipelothrix tonsillarum (Takahashi et al. 1992). Among the genus Erysipelothrix, E. rhusiopathiae is solely

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recognized as the pathogenic species for swine and chicken (Takahashi et al. 1987, 1992, 1996), and therefore, species-specific identification of E. rhusiopathiae is crucial. Differentiation of the two species using biochemical characteristics is time-consuming and laborious. A rapid, simple and practical assay for the identification of E. rhusiopathiae has been sought. Several PCR assays offer a more sophisticated approach to the identification of E. rhusiopathiae (Shimoji et al. 1998; Takeshi et al. 1999). Although PCR assays provide more rapid identification of E. rhusiopathiae than conventional biochemical-based assays, they require the use of electrophoresis to detect amplified products, which is time-consuming and tedious. A recent real-time PCR assay for the identification of E. rhusiopathiae (Pal et al.2010) is more rapid than conventional PCR assays, but requires sophisticated and expensive equipment. A recently developed novel nucleic acid amplification method termed loop-mediated isothermal amplification (LAMP) (Notomi et al. 2000) is a promising candidate for the rapid and easy detection of E. rhusiopathiae. This LAMP assay allows one-step detection of gene amplification by a simple turbidity analysis and requires only a simple incubator, such as a heat block or a water bath providing a constant temperature. LAMP assays are faster, easier to perform and more specific than conventional PCR assays. Further, because they synthesize a large amount of DNA and its by-product, an insoluble white precipitate of magnesium pyrophosphate, the by-product can be detected by a simple turbidity analysis. The increase in turbidity of the reaction mixture due to the production of the white precipitate correlates with the amount of DNA synthesized (Notomi et al. 2000). Thus, compared to real-time PCR assays, expensive equipment is not necessary to obtain a high level of precision. In addition, there are fewer preparation steps for the LAMP assay than for conventional real-time PCR assays, and LAMP assays are less time-consuming in their set-up. These features allow simple, rapid and cost-effective detection of pathogens. Here, we describe a rapid, simple and cost-effective LAMP assay for the detection of E. rhusiopathiae. Further, using 35 swine samples, we attempted to apply this LAMP assay for the rapid screening of E. rhusiopathiae in the enrichment culture broths. Results and discussion The assay developed in this study provided simpler and more rapid detection of E. rhusiopathiae than conventional biochemical- and PCR-based assays. As shown in Table 1, these results were perfectly consistent with those obtained from the conventional PCR assay in our previous study (Shimoji et al. 1998). No cross-reactions were

LAMP for E. rhusiopathiae detection

observed in any of the strains tested, including E. tonsillarum serovar 10 strains, which are difficult to be discriminated from E. rhusiopathiae strains, indicating that the assay is highly species specific. The LAMP screening results were also matched with those obtained with a conventional culture method for detecting E. rhusiopathiae in the 35 enrichment cultures. Using extracted DNA from a single colony, the gelbased PCR assay took 4 h to provide a result, but the LAMP assay was faster, requiring only 17–60 min for amplification and detection of E. rhusiopathiae-specific sequences. All LAMP detections, including the DNA extraction step, were completed in 37–80 min. Representative LAMP results using both the real-time turbidimeter and by visual inspection are shown in Fig. 1a,b. An increase in turbidity due to the LAMP reaction was confirmed using both the real-time turbidimeter and by visual inspection, with all of the results of the two methods showing perfect agreement. Furthermore, the LAMP reaction was also less expensive as described in the literature (Hill et al. 2008). As shown in Table 1, the three published PCR assays were evaluated. The first and third PCR assays for specific detection of E. rhusiopathiae (Shimoji et al. 1998) and E. tonsillarum (Takeshi et al. 1999) successfully identified all 39 E. rhusiopathiae and 12 E. tonsillarum, respectively. However, the second assay for specific detection of E. rhusiopathiae (Takeshi et al. 1999) detected all 39 E. rhusiopathiae, as well as 2 E. tonsillarum serovar 10 strains (Lengyel-P and 2179). The DNA–DNA hybridization test is recognized as the ‘gold standard’ of the classificatory criterion for the bacterial species. Therefore, we judged the two strains as E. tonsillarum, according to the results of the DNA–DNA hybridization test (Takahashi et al. 1992). As shown in Table 2, E. rhusiopathiae was isolated from four of the 35 swine samples by plating; the four samples were also positive with the LAMP assay, but two (skin and vegetation on the heart valve) samples were positive and remaining two (another skin and internal iliac lymph node) samples were negative with the PCR assay we previously developed (Shimoji et al.1998). Remaining 31 E. rhusiopathiae culture-negative samples were all negative with both LAMP and PCR assays. All LAMP results from 35 swine samples using both the realtime turbidimeter and by visual inspection completely matched. Representative LAMP results by the real-time turbidimeter were shown in Fig. 2. Food components, such as organic and phenolic compounds, glycogen, fats and Ca2+, were reported to inhibit DNA polymerase activity (Wilson 1997). PCR assay is more susceptible than the LAMP assay for inhibitory effect (Kaneko et al. 2007). The discrepancy of the two samples showing both

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Table 1 Results of LAMP and PCR assays for the detection of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum PCR results Species

Strain

No. of strains

Serovar

LAMP results

1*

2†

Erysipelothrix rhusiopathiae

ME-7 422/1E R32E11 NF4E1 Doggerscharbe Pecs 67 Tuzok Goda Kaparek 14B IV12/8 Pecs 9 Pecs 3597 Tanzania 545 2017 Bano 36 CJPT-97 MEW 22 GTC 00555T GTC 10359 GTC 10360 GTC 10361 GTC 10362 GTC 10363 GTC 13009 GTC 13010 Field isolates

39

1a 1b 2a 2b 4 5 6 8 9 9 11 12 15 16 17 19 21 23 N 2 ND ND ND ND ND ND ND ND

+ + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + +

Erysipelothrix tonsillarum

Erysipelothrix sp. strain 1 Erysipelothrix sp. strain 2 Non-Erysipelothrix bacteria §

Witlling P-43 Lengyel-P 2179 Iszap-4 2553 Bano 107 CJSF 14-2 KS20A L136 GTC 01527T Field isolate Pecs 56 715

(12) 12

3 7 10 10 14 20 22 24 25 26 7 ND 13 18 ND

(1) 1 1 16

+ +

ND

2†

+ + + + + + + + + + + +

ND

ND, not determined; LAMP, loop-mediated isothermal amplification. *PCR for specific detection of E. rhusiopathiae (Shimoji et al. 1998). †PCR for specific detection of E. rhusiopathiae (Takeshi et al.1999). ‡PCR for specific detection of E. tonsillarum (Takeshi et al.1999). §Details are shown in the Materials and Methods section.

culture positive and LAMP positive but PCR negative may have been, therefore, due to a false negative in the PCR assay caused by inhibition of DNA amplification by components in the swine samples. 364

Using a dilution series of E. rhusiopathiae GTC 00555T in pure cultures and in spiked swine samples, it was shown that the detection limit of the LAMP assay was at least 10-fold superior to that of the conventional PCR

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(a)

(b) 0·7 0·6

Turbidity

0·5 0·4 0·3 0·2 0·1 0 0

10

20

30

40

50

2

1

60

Time (min) Figure 1 Real-time monitoring of loop-mediated isothermal amplification (LAMP) reactions with Erysipelothrix rhusiopathasiae and Erysipelothrix tonsillarum control strains. Representative profiles for qualitative detection are shown. (a) The total DNA extracted from E. rhusiopathasiae (■, GTC 00555T; ●, GTC 10359; ▲, ME-7; ○, MEW 22), and E. tonsillarum (□, GTC 01527T) was examined. (b) Visual detection by observation of precipitation: tube 1, E. rhusiopathasiae GTC 00555T; tube 2, E. tonsillarum GTC 01527T.

Table 2 Comparison between LAMP, PCR and Erysipelothrix rhusiopathiae isolation results in 35 swine samples LAMP results

PCR results*

Culture results

Positive

Negative

Positive

Negative

Positive (n = 4) Negative (n = 31)

4 0

0 31

2 0

2 31

LAMP, loop-mediated isothermal amplification. *PCR for specific detection of E. rhusiopathiae (Shimoji et al. 1998).

0·6 0·5 0·4 Turbidity

assay (Table 3). The LAMP assay was able to detect E. rhusiopathiae as low as 12 CFU per test tube in pure cultures and in spiked swine samples. DNA amplification in the LAMP assay could also be judged as white precipitate by visual assessment with the unaided eye. In the LAMP assay, the detection limits determined by the two judgement methods were the same. The detection limit of the LAMP assay is 10- to 100-fold superior to conventional PCR assay and typically equivalent to real-time PCR assay (Yamazaki et al. 2009). We developed a novel LAMP assay for the detection of E. rhusiopathiae. An E. rhusiopathiae primer set based on the putative polypeptide gene (GenBank accession number, AP012027, ERH_0856), which is involved in the biosynthesis of capsular polysaccharides (Shimoji et al.1998; Ogawa et al. 2011; Shi et al. 2012), was designed as the sequence has proven to be highly conserved and specific for E. rhusiopathiae (Shimoji et al.1998). Further, using BLAST research (http://blast.ncbi.nlm.nih.gov/Blast.cgi), we have confirmed specificity of the LAMP primers. While all of the six primers perfectly matched E. rhusiopathiae target sequence (AP012027, ERH_0856), the primer set recognizing regions in the ERH_0856 (910 217–910 423, 207 bp) did not match any bacterial sequence in the GenBank (http://www.ncbi.nlm.nih.gov/genbank). The result reinforces the high specificity of the LAMP primers we developed. The frequent worldwide swine erysipelas occurrence and consequently economic losses to livestock industries highlight the need for control of swine erysipelas. Isothermal amplification methods such as LAMP are now being applied for the routine detection of pathogens that impact both human and veterinary health. As the specific amplification occurs at a constant temperature, there is less

0·3 0·2 0·1 0 0

10

20

30

40

50

60

Time (min) Figure 2 Real-time monitoring of Erysipelothrix rhusiopathasiaespecific loop-mediated isothermal amplification (LAMP) reactions with extracted DNAs from enrichment broth cultures. Representative profiles for qualitative detection are shown. The total DNA extracted from culture-positive samples (M, skin; s, internal iliac lymph node) and culture-negative samples (s, synovial fluid; ●, vegetation on the heart valve) was examined.

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Table 3 Detection limits of the LAMP and PCR assays for Erysipelothrix rhusiopathiae

PCR assays previously published (Shimoji et al.1998; Takeshi et al.1999).

Dilutions of cultures for assays 10 Pure cultures Cells per test tube LAMP PCR* Spiked swine samples Cells per test tube LAMP PCR*

1

10

2

10

3

10

12037 ND +

1203 + +

120 + +†

12 +

12037 ND +

1203 + 

120 +

12 +

4

10

5

10

6

01

001

ND

ND

01

001

ND

ND

ND, not determined; +, duplicate assay: both positive; , duplicate assay: one negative and one positive; , duplicate assay: both negative; LAMP, loop-mediated isothermal amplification. *PCR for specific detection of E. rhusiopathiae (Shimoji et al. 1998). †Faint bands at the predicted size (937 bp) were observed.

reliance upon expensive equipment and there is obvious potential to use of these assays as the basis of an inexpensive molecular test. Furthermore, high sensitivity of our LAMP assay and the fact that the amplification reaction results can be judged by simple visual assessment make it a more attractive tool for routine diagnosis. Application of the LAMP assay for rapid screening from clinical samples would be time-saving and cost-effective (Yamazaki 2013). Further study is required to apply the LAMP assay for direct detection of E. rhusiopathiae from animal specimens, which may enable early diagnosis (Pal et al.2010). The LAMP assay presented here may potentially facilitate surveillance and early diagnosis of swine erysipelas in the field. Materials and methods

Non-Erysipelothrix rhusiopathiae strains Of the 30 non-E rhusiopathiae strains, 12 strains were Erysipelothrix tonsillarum, comprising of GTC 01527T, 10 serovar reference strains from NIAH and one isolate from a swine sample at the slaughterhouse in Kumamoto, Japan, in 2012. Remaining 18 strains were comprised of two serovar reference strains of Erysipelothrix sp., six isolates of Streptococcus suis and one isolate each of Arcanobacterium pyogenes, Campylobacter jejuni, Enterobacter faecalis, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella Choleraesuis, Salmonella Infantis, Streptococcus pneumoniae and Streptococcus sp. Culture conditions All Erysipelothrix strains were cultured on blood agar (Oxoid Ltd, Basingstoke, Hampshire, England) supplemented with 5% (v/v) lysed sheep blood at 37°C for 18–24 h in aerobic conditions. Camp. jejuni was incubated at 37°C for 48 h in a microaerobic atmosphere. Other bacterial strains were cultured on blood agar or nutrient agar (Nissui Pharmaceutical Co. Ltd, Tokyo, Japan) at 37°C for 20–24 h in aerobic conditions. DNA extraction from culture Bacterial DNA was extracted from all of the 69 strains tested, according to the method of Galan and Timoney (1990) or the method as previously described (Yamazaki 2013). The resulting supernatant was stored at 20°C until use, at which time 1 ll samples were used as template DNA for the LAMP and PCR assays.

Bacterial strains

Primer design for the LAMP assay

Erysipelothrix rhusiopathiae strains All of the 69 bacterial strains used are listed in Table 1. Of the 39 E. rhusiopathiae strains, eight E. rhusiopathiae reference strains were obtained from Gifu Type Culture Collection (Gifu, Japan): GTC 00555T, GTC 10359, GTC 10360, GTC 10361, GTC 10362, GTC 10363, GTC 13009 and GTC 13010. Superscript T designates a type strain. Other 19 strains were serovar reference strains from National Institute of Animal Health (NIAH), Japan. Remaining 12 isolates were obtained from swine samples at the slaughterhouses in Miyazaki, Japan, between 2012 and 2013. The 12 isolates were identified by their Gram stain appearance (Erysipelothrix isolates are Gram positive and appear to be rods) and the API Coryne system (BioMerieux S.A., Marcy l’Etoile, France), as well as by the

All primers recognizing eight distinct regions of the putative polypeptide gene, which codes for a capsular polysaccharide biosynthesis and is a specific and conserved sequence for E. rhusiopathiae (Shimoji et al.1998; Ogawa et al. 2011; Shi et al. 2012), were designed using Primer Explorer V4 software (Fujitsu System Solutions, Tokyo, Japan). The primers were synthesized in sequence-grade purification by Hokkaido System Science Co., Ltd. (Sapporo, Japan). Details of each primer designed are shown in Table 4. Primer FIP consisted of the F1 complementary sequence and the F2 sequence. Primer BIP consisted of the B1 direct sequence and the B2 complementary sequence. Primers B3 and LF consisted of the B3 and LF complementary sequences, respectively.

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Table 4 LAMP primers used Primer designation

Sequence (5′–3′)

Gene location

ER-FIP ER-BIP ER-F3 ER-B3 ER-LF ER-LB

ACGTCGAACTGTTAGATATGCAAAAGATTTAACACAATTTTCAATCGTCC TTGTAATGATGAGCGCGATGAGGGAAAGATTCTCTTGATACTTTGA ATCGTGGACCAAACAAGC CTGATTCATGATCCCACATTG CGATTGTCATAATCATCATAGCTC CTTTTAGATTCTCAAGCCGCGTTA

910 910 910 910 910 910

314–910 317–910 217–910 423–910 289–910 339–910

290, 910 241–910 265 338, 910 395–910 372 234 403 266 362

All primers were designed from the putative polypeptide gene sequence of E. rhusiopathiae AP012027, ERH_0856, deposited to the GenBank (Shimoji et al.1998; Ogawa et al. 2011). LAMP, loop-mediated isothermal amplification.

LAMP assay The LAMP assay was performed using single 25 ll reaction mixtures containing 1 ll of template DNA, eight units of Bst DNA polymerase (large fragment; New England Biolabs, Ipswich, MA), 20 mmol l 1 Tris-HCl (pH88, Wako Pure Chemical Industries, Osaka, Japan), 10 mmol l 1 KCl (Sigma-Aldrich, St. Louis, MI), 8 mmol l 1 MgSO4 (New England Biolabs), 10 mmol l 1 (NH4) 2SO4 (Sigma-Aldrich), 01% Tween20 (SigmaAldrich), 08 mol l 1 Betaine (Sigma-Aldrich), 1 14 mmol l each dNTPs (GE Healthcare, Little Chalfont, UK), 16 lmol l 1 each of inner primers FIP and BIP; 02 lmol l 1 each of outer primers F3 and B3; and 08 lmol l 1 each of loop primers LF and LB. The reaction mixtures were incubated at 65°C for 60 min, followed by 80°C for 5 min to complete the reaction using a realtime turbidimeter (Loopamp EXIA; Teramecs, Kyoto, Japan). Amplified products were detected at 650 nm using a Loopamp EXIA turbidimeter. A reaction was considered positive when the turbidity reached 01 within 60 min. PCR assays The PCR assays were performed using single 50 ll reaction mixtures containing 1 ll of template DNA. An E. rhusiopathiae-specific PCR assay was carried out using the primers ER1 (5′- CGA TTA TAT TCT TAG CAC GCA ACG -3′) and ER2 (5′- TGC TTG TGT TGT GAT TTC TTG ACG -3′), as described in our previous study (Shimoji et al.1998). Second and third PCR assays were performed to correctly differentiate the two closely related species at the genetic level using two sets of the primers, ER1-F(5′GTT CAT CTC TCT AAT GCA CTA C -3′) and ER1-R (5′- TGT TGG ACT ACT AAT CGT TTC G -3′) for E. rhusiopathiae, and ER2-F (5′- ATG TAA TAT GAT CTG GTG ATT TG -3′) and ER2-R (5′- AGG ACT GCT GAT TGT CTC ATG -3′) for E. tonsillarum, as described in a published paper (Takeshi et al.1999). Using E. rhusiopathiae GTC 00555T and E. tonsillarum GTC 01527T as

positive and negative controls, the evaluation of the LAMP and PCR assays was performed in a blind fashion. Isolation of Erysipelothrix rhusiopathiae from swine sample A total of 35 swine samples obtained at the slaughterhouses in Miyazaki prefecture, Japan, were used; these samples included 19 vegetation on the heart valves, 13 skins, two synovial fluids and one internal iliac lymph node. Approximately 2 g of the swine samples were added to 10 ml of tryptic soy broth (Becton Dickinson and Co., Sparks, MD) containing 0005% gentamicin (Schering-Plough, Kenilworth, NJ) and 01% tween 80 (Yoneyama Yakuhin Kogyo Co., Ltd., Osaka, Japan) (GT-TSB) and were followed by thorough mixing using a Vortex-Genie 2 (Scientific Industries Inc., New York, NY). Samples were then incubated at 37°C for 18–20 h. For E. rhusiopathiae isolation, one loopful of a GT-TSB broth culture sample was streaked onto blood agar using a disposable loop, which was followed by incubation at 37°C for 18–72 h. Among the 35 swine samples, typical Erysipelothrix-like colonies were isolated from four samples, comprising two skins and one each of internal iliac lymph node and vegetation on the heart valve. The Erysipelothrix-like colonies on the blood agar were identified as described above. DNA extraction from enrichment broth Erysipelothrix rhusiopathiae detection was performed by LAMP and PCR assays using 1 ml of each GT-TSB enrichment culture sample, which was used for preparation of DNA templates. To remove larger debris in the swine sample and components of the GT-TSB enrichment broth, a GT-TSB enrichment culture was thoroughly mixed and centrifuged at 900 g for 1 min. The resulting supernatant was transferred to a new 15-ml microcentrifuge tube and centrifuged at 10 000 g for 5 min. After removal of the supernatant, pellets were resuspended in 50 ll of NaOH (25 mmol l 1), thoroughly mixed using a

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Vortex-Genie 2 and heated at 95°C for 10 min. After neutralization with 4 ll of Tris-HCl buffer (1 mol l 1, pH 75), debris was centrifuged at 4°C and 15 000 g for 5 min. Supernatants were stored at 20°C until use, at which time 1 ll samples were used as template DNA for both LAMP and PCR assays. Comparison for detection limits of the LAMP and PCR assays Detection limits of the LAMP and PCR assays for E. rhusiopathiae both in pure cultures and in artificially spiked swine samples were determined using E. rhusiopathiae GTC 00555T. Serial 10-fold dilutions were prepared in PBS (phosphate-buffered saline) using fresh pure cultures incubated at 37°C for 14 h on blood agar. In parallel, to determine the inoculated size, 100 ll of each dilution was plated onto blood agar and incubated at 37°C for 72 h. For preparation of the template DNA from pure cultures, 100 ll of each was transferred to a 15-ml microcentrifuge tube and was centrifuged for 5 min at 15 000 g. After removal of the supernatant, the pellets were suspended in 50 ll of NaOH (25 mmol l 1), and the mixture was heated at 95°C for 10 min. After neutralization with 4 ll of Tris-HCl buffer (1 mol l 1, pH 75), debris was pelleted by centrifugation at 15 000 g, 4°C for 5 min. Then 1 ll of each supernatant was used as DNA template. For preparation of the template DNA from spiked swine samples, 10 ml of GT-TSB enrichment broth containing 2 g of the E. rhusiopathiae-negative vegetation on the heart valve was incubated at 37°C for 20 h. Using the serial 10-fold bacterial dilutions described above, 100 ll of each was spiked into 900 ll of the GT-TSB enrichment broth culture in a 15-ml microcentrifuge tube and then mixed well using a Vortex-Genie 2. Template DNA was concentrated at 54 ll of supernatant as mentioned above, and 1 ll of each was used as DNA template. The detection limit tests of the LAMP assay were conducted in duplicate, and the detection limits were defined as the last positive dilutions, with the sample considered positive if both two samples tested positive. Conflict of interest We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. References Galan, J.E. and Timoney, J.F. (1990) Cloning and expression in Escherichia coli of a protective antigen of Erysipelothrix rhusiopathiae. Infect Immun 58, 3116–3121. 368

Hill, J., Beriwal, S., Chandra, I., Paul, V.K., Kapil, A., Singh, T., Wadowsky, R.M., Singh, V. et al. (2008) Loopmediated isothermal amplification assay for rapid detection of common strains of Escherichia coli. J Clin Microbiol 46, 2800–2804. Kaneko, H., Kawana, T., Fukushima, E. and Suzutani, T. (2007) Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods 70, 499–501. Makino, S., Okada, Y., Maruyama, T., Ishikawa, K., Takahashi, T., Nakamura, M., Ezaki, T. and Morita, H. (1994) Direct and rapid detection of Erysipelothrix rhusiopathiae DNA in animals by PCR. J Clin Microbiol 32, 1526–1531. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T. (2000) Loopmediated isothermal amplification of DNA. Nucleic Acids Res 28, E63. Ogawa, Y., Ooka, T., Shi, F., Ogura, Y., Nakayama, K., Hayashi, T. and Shimoji, Y. (2011) The genome of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, reveals new insights into the evolution of firmicutes and the organism’s intracellular adaptations. J Bacteriol 193, 2959–2971. Pal, N., Bender, J.S. and Opriessnig, T. (2010) Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real-time PCR assay. J Appl Microbiol 108, 1083–1093. Shi, F., Harada, T., Ogawa, Y., Ono, H., Ohnishi-Kameyama, M., Miyamoto, T., Eguchi, M. and Shimoji, Y. (2012) Capsular polysaccharide of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, and its modification with phosphorylcholine. Infect Immun 80, 3993–4003. Shimoji, Y. (2000) Pathogenicity of Erysipelothrix rhusiopathiae: virulence factors and protective immunity. Microbes Infect 2, 965–972. Shimoji, Y., Mori, Y., Hyakutake, K., Sekizaki, T. and Yokomizo, Y. (1998) Use of an enrichment broth cultivation-PCR combination assay for rapid diagnosis of swine erysipelas. J Clin Microbiol 36, 86–89. Takahashi, T., Sawada, T., Muramatsu, M., Tamura, Y., Fujisawa, T., Benno, Y. and Mitsuoka, T. (1987) Serotype, antimicrobial susceptibility, and pathogenicity of Erysipelothrix rhusiopathiae isolates from tonsils of apparently healthy slaughter pigs. J Clin Microbiol 25, 536–539. Takahashi, T., Fujisawa, T., Tamura, Y., Suzuki, S., Muramatsu, M., Sawada, T., Benno, Y. and Mitsuoka, T. (1992) DNA relatedness among Erysipelothrix rhusiopathiae strains representing all twenty-three serovars and Erysipelothrix tonsillarum. Int J Syst Bacteriol 42, 469–473. Takahashi, T., Nagamine, N., Kijima, M., Suzuki, S., Takagi, M., Tamura, Y., Nakamura, M., Muramatsu, M. et al. (1996) Serovars of Erysipelothrix strains isolated from pigs affected with erysipelas in Japan. J Vet Med Sci 58, 587–589.

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LAMP for E. rhusiopathiae detection

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Letters in Applied Microbiology 58, 362--369 © 2013 The Society for Applied Microbiology

369

Development of a loop-mediated isothermal amplification assay for rapid and simple detection of Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae is a causative agent of swine erysipelas. We developed a novel and highly specific loop-mediated isothermal amplification...
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