In this study， the specific mapA gene was used to design a set of primers to develop a loop-mediated isothermal amplification ( LAMP) assay for detection of C． jejuni． To optimize the LAMP assay， the reaction components in the assay were screened to determine the optimal concentration． The final LAMP assay comprised 1．6μmol /L each of inner primers FIP and BIP，0．2 μmol /L each of outer primers F3 and B3,8 mmol /L Mg2 + ，1．4mmol /L dNTP，0 ．8 mol /L Betaine． The specificity test showed that the assay correctly identified all C． jejuni strains but not 13 other bacterial species． The sensitivity of the assay was 100 fg per test tube for C． jejuni genomic DNA and 7． 5 cfu per test tube for C． jejuni bacterial culture． This LAMP assay is a rapid and simple tool for detection of C． jejuni and will provide an essential role in facilitating early diagnosis of this organism from animal products．
Xu Zijian
,
Yang Bing
,
Su Xia
,
Zhou Hongzhuan
,
Shi Aihua
,
Xu Fuzhou
. Development and Optimization of a Loop-mediated Isothermal Amplification Assay for Detection of Campylobacter jejuni[J]. Acta Agriculturae Boreali-Sinica, 2013
, 28(3)
: 222
-226
.
DOI: 10.3969/j.issn.1000-7091.2013.03.040
[1] Snelling W J，Matsuda M，Moore J E，et al． Campylobacter jejuni[J]． Lett Appl Microbiol，2005，41( 4) :297 - 302．
[2] Lin J． Novel approaches for Campylobacter control in poultry[J]． Foodborne Pathog Dis，2009，6( 7) : 755 - 765．
[3] Poly F，Guerry P． Pathogenesis of Campylobacter[J]．Curr Opin Gastroenterol，2008，24( 1) : 27 - 31．
[4] Jagusztyn-Krynicka E K，Laniewski P，Wyszynska A．Update on Campylobacter jejuni vaccine development for preventing human campylobacteriosis[J]． Expert Rev Vaccines，2009，8( 5) : 625 - 645．
[5] Chen X，Naren G W，Wu C M，et al． Prevalence and antimicrobial resistance of Campylobacter isolates in broilers from China[J]． Vet Microbiol，2010，144( 1 -2) : 133 - 139．
[6] Denis M，Soumet C，Rivoal K，et al． Development of am-PCR assay for simultaneous identification of Campylobacter jejuni and C． coli[J]． Lett Appl Microbiol，1999，29( 6) : 406 - 410．
[7] Notomi T，Okayama H，Masubuchi H，et al． Loop-mediated isothermal amplification of DNA[J]． Nucleic Acids Res，2000，28( 12) : E63．
[8] Mori Y，Notomi T． Loop-mediated isothermal amplification ( LAMP) : a rapid，accurate，and cost-effective diagnostic method for infectious diseases[J]． J Infect Chemother，2009，15( 2) : 62 - 69．
[9] Parida M，Sannarangaiah S，Dash P K，et al． Loop mediated isothermal amplification ( LAMP) : a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases[J]． Rev Med Virol，2008，18( 6) : 407 - 421．
[10] Luo Y，Sahin O，Dai L，et al． Development of a loopmediated isothermal amplification assay for rapid，sensitive and specific detection of a Campylobacter jejuni clone[J]． J Vet Med Sci，2012，74( 5) : 591 - 596．
[11] Yamazaki W，Taguchi M，Ishibashi M，et al． Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli[J]． J Med Microbiol，2008，57( Pt 4) : 444 - 451．
[12] Yamazaki W，Taguchi M，Ishibashi M，et al． Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Campylobacter fetus[J]． Vet Microbiol，2009，136( 3 - 4) : 393 - 396．
[13] Yamazaki W，Taguchi M，Kawai T，et al． Comparison of loop-mediated isothermal amplification assay and conventional culture methods for detection of Campylobacter jejuni and Campylobacter coli in naturally contaminated chicken meat samples[J]． Appl Environ Microbiol，2009，75( 6) : 1597 - 1603．
[14] Stucki U，Frey J，Nicolet J，et al． Identification of Campylobacter jejuni on the basis of a species-specific gene that encodes a membrane protein[J]． J Clin Microbiol，1995，33( 4) : 855 - 859．
[15] Parkhill J，Wren B W，Mungall K，et al． The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences[J]． Nature，2000，403( 6770) : 665 - 668．
[16] 黄金林． 我国部分地区鸡源空肠弯曲菌流行病学及运送flaA 基因的壳聚糖纳米DNA 疫苗免疫生物学特性研究[D]． 扬州: 扬州大学，2008．
