Application of a multiplex PCR assay for identification of Campylobacter fetus and subspecies differentiation in fecal samples from reptile animals
-
摘要:
目的 建立一种检测胎儿弯曲菌胎儿亚种、性病亚种和龟亚种的多重PCR检测方法。 方法 使用针对胎儿弯曲菌、性病亚种和龟亚种的特异性引物,优化反应体系及反应条件,使用38株菌株(18株胎儿弯曲菌和20株非胎儿弯曲菌)验证其特异性,并将该方法应用于53份爬行动物粪便筛查。 结果 针对3种亚种均可扩增相应的片段,胎儿亚种只有1条359 bp大小的条带、性病亚种有2条分别为359 bp和156 bp大小的条带,龟亚种有2条分别为359 bp和266 bp大小的条带。优化后的最佳扩增条件为:退火温度58 ℃,引物MG3f和Cf359r浓度为0.1 μM,ISC2mf和ISC2mr、CoA266f和CoA266r的浓度0.2 μM。对胎儿亚种、性病亚种和龟亚种的检出限分别为0.40 ng/μL、0.39 ng/μL和0.47 ng/μL。使用18株胎儿弯曲菌和20株非胎儿弯曲菌其特异性为100%。对53份爬行动物粪便进行筛查中有1份龟亚种阳性并分离出了相应的菌株。 结论 本研究建立的多重PCR方法能利用1个反应体系同时鉴别3种亚种,从而为菌株快速鉴定、流行病学调查和溯源研究提供技术支持。 Abstract:Objective To establish a multiplex polymerase chain reaction(PCR) method to differentiate C fetus subsp fetus(cff),C fetus subsp venerealis(cfv) and C fetus subsp testudinum(cft). Method The specific primers targetting C fetus,cfv and cft were designed and the reaction conditions were optimized. The multiplex PCR system was also evaluated by using 38 strains (including 18 Campylobacter fetus strains and 20 non-Campylobacter fetus strains) and further applied to 53 fecal samples from reptile animals. Results The multiplex PCR assay with 3 primer pairs could yield specific fragments: only one fragment of 359 bp for cff, two fragments of 359 bp and 156bp for cfv, two fragments of 359 bp and 266 bp for cft. The amplification conditions were also optimized and the extending temperature was 58 ℃. The optimal concentration of primers “MG3f and Cf359r”, “ISC2mf and ISC2mr”,“CoA266f and CoA266r” were 0.1 μM , 0.2 μM and 0.2 μM respectively. The sensitivity of the assay for detecting cff, cfv and cft was 0.40 ng/μL、0.39 ng/μL and 0.47 ng/μL respectively. The analytical specificity was 100% by examination of 18 Campylobacter fetus strains and 20 non-Campylobacter fetus strains. In 53 fecal samples from reptile animals, one was PCR postive for cft and the suspective colonies were also identified as cft. Conclusion The established multiplex PCR assay can differentiate three subspecies simutaneously and provide technical support for rapid identification, epidemiological investigation and tracing infection source. -
Key words:
- Campylobaccter fetus /
- multiplex PCR /
- detection method /
- MALDI-TOF MS
-
图 1 PCR引物验证电泳图。(M为marker;lane1、2、3、4为以MG3f和Cf359r为引物,以胎儿亚种、性病亚种、龟亚种、阴性对照为模板的电泳图;lane5、6为以ISC2mf和ISC2mr为引物,以性病亚种、阴性对照为模板的电泳图;lane7、8为以Coa266f和Coa266r为引物,龟亚种、阴性对照为模板的电泳图)
Figure 1. PCR results with three pairs of primers. Lane M: marker; lane1−4: cff, cfv, cft and negative control, "MG3f and Cf359r" as primers; lane 5−6: cfv and negative control, "ISC2mf and ISC2mr" as primers; lane7−8:cft and negative control, "Coa266f and Coa266r" as primers.
图 2 退火温度在56 ℃,58 ℃,60 ℃和62 ℃时三种亚种标准菌株的PCR产物电泳图。(M为marker;lane1,4,7,10为胎儿亚种;lane2,5,8,11为性病亚种;lane3,6,9,12为龟亚种)
Figure 2. Multiplex PCR results for identification of three subspecies at four different annealing temperature (56 ℃, 58 ℃, 60 ℃ and 62 ℃). (M: marker; lane1, 4, 7, 10: cff; lane2, 5, 8, 11:cfv; lane3, 6, 9, 12: cft)
图 3 引物在不同浓度比例的PCR产物电泳图。(M为marker;lane1-3为MG3f和Cf359r浓度0.2 μM,lane4-6为MG3f和Cf359r浓度0.1μM,lane7-9为MG3f和Cf359r浓度0.05μM)
Figure 3. Multiplex PCR results at different relative concentration of primers. (M: marker; lane1−3: The concentration of MG3f and Cf359r is 0.2 μM, lane4−6: The concentration of MG3f and Cf359r is 0.1μM, lane7−9: The concentration of MG3f and Cf359r is 0.05 μM)
图 4 多重PCR方法对胎儿亚种、性病亚种和龟亚种的灵敏度实验结果。(lane1-4:胎儿亚种DNA浓度为40 ng/μL、4 ng/μL、0.4 ng/μL和0.04 g/μL;lane5-8:性病亚种DNA浓度为39 ng/μL、3.9 ng/μL、0.39 ng/μL和0.04 g/μL;龟亚种DNA浓度为47 ng/μL、4.7 ng/μL、0.47 ng/μL和0.05 ng/μL)
Figure 4. The sensitivity of the multiplex PCR for detecting cff, cfv and cft. (lane1−4: the concentration of cff is 40 ng/μL、4 ng/μL、0.4 ng/μL and 0.04 g/μL; lane 5−8: the concentration of cfv is 39 ng/μL、3.9 ng/μL、0.39 ng/μL and 0.04 g/μL; lane 9−12: the concentration of cft is 47 ng/μL、4.7 ng/μL、0.47 ng/μL and 0.05 ng/μL).
图 5 多重PCR特异性实验结果。(M:marker;lane1-2:胎儿弯曲菌胎儿亚种;lane3-4:胎儿弯曲菌性病亚种;lane5-18:胎儿弯曲菌龟亚种;lane19:空肠弯曲菌;lane20:大肠弯曲菌;lane21:海鸟弯曲菌;lane22:大肠埃希氏;lane23:沙门菌; lane24:志贺菌; lane25:变形杆菌; lane26:霍乱弧菌; lane27:副溶血性弧菌; lane28:创伤弧菌;lane29:铜绿假单胞菌; lane30:金黄色葡萄球菌; lane31:脑膜炎奈瑟菌; lane32:猪链球菌; lane33:溶血性链球菌; lane34:柠檬酸杆菌; lane35:粪产碱杆菌; lane36:嗜水气单胞菌; lane37:粘质沙雷菌; lane38:蜡样芽孢杆菌)
Figure 5. The specifity results of the multiplex PCR assay. (M:marker; lane1−2: cff; lane3−4: cfv; lane5−18: cft; lane19: Campylobacter jejuni; lane20:Campylobacter coli; lane21: Campylobacter lari; lane22:E.coli; lane23:Salmonella; lane24:Shigella; lane25:Proteus ; lane26: Vibrio cholerae; lane27:Vibrio parahaemolyticus; lane28:Vibrio vulnificus; lane29:Pseudomonas aeruginosa; lane30:Staphylococcus aureus; lane31:Neisseria meningitidis; lane32:Streptococcus suis; lane33:Hemolytic streptococci; lane34:Citrobacter; lane35: Alcaligenes faecalis; lane36: Aeromonas hydrophila; lane37:Serratia marcescens; lane38:Bacillus cereus)
表 1 多重PCR引物序列及扩增产物大小
Table 1. The nucleotide sequences of primer sets and the size of the multiplex PCR products
-
[1] Fitzgerald C, Tu Z C, Patrick M, et al. Campylobacter fetus subsp. testudinum subsp. nov. , isolated from humans and reptiles[J]. Int J Syst Evol Microbiol, 2014, 64(Pt 9): 2944–2948. DOI: 10.1099/ijs.0.057778−0. [2] 侯水平, 陈守义. 胎儿弯曲菌的感染现状、检测方法和分子分型的研究进展[J]. 中国人兽共患病学报,2014,30(1):85–88. DOI:10.3969/cjz.j.issn.1002−2694.2014.01.018.Hou SP, Chen SY. Research progress on the infection status, testing methods and molecular subtyping of Campylobacter fetus[J]. Chinese J of Zoonoses, 2014, 30(1): 85–88. DOI: 10.3969/cjz.j.issn.1002−2694.2014.01.018. [3] Yamazaki-Matsune W, Taguchi M, Seto K, et al. Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis[J]. J Med Microbiol, 2007, 56(Pt 11): 1473. DOI: 10.1099/jmm.0.47363−0. [4] van der Graaf-van B L, van Bergen M A, van der Wal F J, et al. Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay[J]. J Microbiol Methods, 2013, 95(1): 93–97. DOI: 10.1016/j.mimet.2013.06.005. [5] Steele T W, McDermott S N. The use of membrane filters applied directly to the surface of agar plates for the isolation of Campylobacter jejuni from feces[J]. Pathology, 1984, 16(3): 263–265. DOI: 10.3109/00313028409068535. [6] 屈平华, 赵红波, 黄彬, 等. 卫生部微生物室间质评1012菌的鉴定与放线菌属的系统发生分析[J]. 中华检验医学杂志,2011,34(9):814–819. DOI:10.3760/cma.j.issn.1009−9158.2011.09.010.Qu PH, Zhao HB, Huang B, et, al. Identification of the strain 1012 isolated from the sample of EQA of the Chinese National Center of Clinical Laboratory, and phylogenetic analysis of Actinomyces turicensisand related bacteria in the family Actinomycetaceae[J]. Chinese J of Lab Med, 2011, 34(9): 814–819. DOI: 10.3760/cma.j.issn.1009−9158.2011.09.010. [7] Health W O F A. OIE-Listed diseases, infections and infestations, in force in 2021[EB/OL]. (2021−01−31)[1月31日].https://www.oie.int/en/animal-health-in-the-world/oie-listed-diseases-2021/. [8] Mshelia G D, Amin J D, Woldehiwet Z, et al. Epidemiology of bovine venereal Campylobacteriosis: geographic distribution and recent advances in molecular diagnostic techniques[J]. Reprod Domest Anim, 2010, 45(5): e221–e230. DOI: 10.1111/j.1439−0531.2009.01546.x. [9] 朱战波, 崔玉东, 侯喜林, 等. 奶牛流产胎儿弯曲菌的分离鉴定[J]. 中国兽医杂志,2004,40(12):28–30. DOI:10.3969/j.issn.0529−6005.2004.12.011.Zhu ZB, Cui YD, Hou XL, et, al. Isolation and identification ofCampylobacter fetus from the abortive cow[J]. Chinese J of Vet Med, 2004, 40(12): 28–30. DOI: 10.3969/j.issn.0529−6005.2004.12.011. [10] 赵红琼. 新疆奶牛子宫内膜炎病原菌的分离鉴定及其治疗中草药的筛选[D]. 新疆农业大学, 2005.Zhao HQ. Isolation and Identification of Pathogens of Bovine Endometritis in Xinjiang and Screening for Herbs for Its Treatment[D]. Xinjiang Agricultural University, 2005 [11] Wang C M, Shia W Y, Jhou Y J, et al. Occurrence and molecular characterization of reptilian Campylobacter fetus strains isolated in Taiwan[J]. Vet Microbiol, 2013, 164(1-2): 67–76. DOI: 10.1016/j.vetmic.2013.01.008. [12] Ledina D, Ivic I, Karanovic J, et al. Campylobacter fetus infection presenting with bacteremia and cellulitis in a 72-year-old man with an implanted pacemaker: a case report[J]. J Med Case Rep, 2012, 6: 414. DOI: 10.1186/1752−1947−6−414. [13] Tanabe S, Kutsuna S, Tsuboi M, et al. Meningoencephalitis Caused by a Campylobacter fetus in a Patient with Chronic Alcoholism[J]. Intern Med, 2019, 58(15): 2247–2250. DOI: 10.2169/internalmedicine.1486−18. [14] Maly M, Willandt B, Nulens E, et al. Campylobacter fetus-induced cellulitis in a Crohn's disease patient under anti-TNFalpha maintenance therapy[J]. Acta Gastroenterol Belg, 2019, 82(2): 335–336. [15] Nishiguchi S, Sekine I, Kuroda S, et al. Myositis Ossificans of the Hip Due to Pyogenic Arthritis Caused by Campylobacter fetus Subspecies fetus[J]. Intern Med, 2017, 56(8): 967–972. DOI: 10.2169/internalmedicine.56.7906. [16] Cunha J S, Queiroz F, Molina R J, et al. Campylobacter fetus spondylodiscitis during immunochemotherapy for non-Hodgkin's lymphoma[J]. Rev Soc Bras Med Trop, 2021: 54. DOI: 10.1590/0037−8682−0801−2020. [17] Nulens E, Decoster E L, Schoonooghe M C, et al. An unexpected Campylobacter fetus infection[J]. Infection, 2018, 46(5): 729–730. DOI: 10.1007/s15010−018−1159−8. [18] Chavez A C, Barrera S, Leon A, et al. Campylobacter fetus Bacteremia in a Healthy Patient Returning from a Trip to the Ecuadorian Amazonia[J]. Zoonoses Public Health, 2017, 64(5): 391–393. DOI: 10.1111/zph.12338. [19] Ishihara A, Hashimoto E, Ishioka H, et al. Campylobacter fetus meningitis associated with eating habits of raw meat and raw liver in a healthy patient: A case report and literature review[J]. IDCases, 2018, 11: 97–100. DOI: 10.1016/j.idcr.2018.02.002. [20] Koppenaal H, Groenendijk F, van den Berge M, et al. [Outbreak of Campylobacter fetus infection after consumption of unpasteurized sheep's milk cheeses: how to trace the source?][J]. Ned Tijdschr Geneeskd, 2017, 161: D1704. [21] 杨小敏, 陈海. 新亚型胎儿弯曲菌引起败血症1例报告[J]. 中国病原生物学杂志, 2008, 3(1): 79. DOI: 10.3969/j.issn.1673-5234.2008.01.032.Yang XM, Chen H. A new septicemia of Campylobacter fetus. J of Pathogen Bio, 2008, 3(1): 79. DOI: 10.3969/j.issn.1673-5234.2008.01.032. [22] 黄健云, 陈光辉, 杨新怀. 血液中两株胎儿弯曲菌胎儿亚种的鉴定、药敏试验及序列分析[J]. 中华检验医学杂志,2012,35(8):753–755. DOI:10.3760/cma.j.issn.1009−9158.2012.08.022.Huang JY, Chen GH, Yang XH. the identification, antimicrobiol resistance and sequence analysis of two Campylobacter fetus strains isolated from blood[J]. Chin J Lab Med, 2012, 35(8): 753–755. DOI: 10.3760/cma.j.issn.1009−9158.2012.08.022. [23] 李春辉, 孙可歆, 孙风辉. 胎儿弯曲菌蛟河株从结肠癌患者血液中的分离及鉴定[J]. 世界华人消化杂志,2010,18(13):1395–1399. DOI:10.3969/j.issn.1009−3079.2010.13.018.Li CH, Sun KQ, Sun FH. Characterization of a novel strain of Campylobacter fetus separated from the blood sample from a patient with colon cancer[J]. World Chin J of Digestology, 2010, 18(13): 1395–1399. DOI: 10.3969/j.issn.1009−3079.2010.13.018. [24] 李倩, 赵玉, 王义义, 等. 胚胎弯曲杆菌所致脑膜炎一例分析并文献复习[J]. 中华神经科杂志,2017,50(12):927–930. DOI:10.3760/cma.j.issn.1006−7876.2017.12.010.Li Q, Zhao Y, Wang YY, et, al. Campylobacter fetus meningitis in adults: report of one case and review of the literature[J]. Chin J of Neurology, 2017, 50(12): 927–930. DOI: 10.3760/cma.j.issn.1006−7876.2017.12.010. [25] 邱伟波, 屈平华, 秦珏. 腹透水中胎儿弯曲菌龟亚种的鉴定及特征分析[J]. 海南医学,2018,29(3):432–433. DOI:10.3969/j.issn.1003−6350.2018.03.042.Qiu WB, Qu PH, Qin Y. the identification and charateristic analysis of Campylobacter fetus subsp. testudinum from Peritoneal dialysate[J]. Hainan Med J, 2018, 29(3): 432–433. DOI: 10.3969/j.issn.1003−6350.2018.03.042. [26] 强济香, 金春梅, 陈丽婷, 等. 胎儿弯曲菌胎儿亚种菌血症一例[J]. 现代医学,2020,48(12):1617–1620. DOI:10.3969/j.issn.1671−7562.2020.12.025.Qiang JX, Jin CM, Chen LT, et, al. A case report: Campylobacter fetus subsp. fetus bacteremia[J]. Modern Med J, 2020, 48(12): 1617–1620. DOI: 10.3969/j.issn.1671−7562.2020.12.025. [27] van Samkar A, Brouwer M C, van der Ende A, et al. Campylobacter fetus Meningitis in Adults: Report of 2 Cases and Review of the Literature[J]. Medicine (Baltimore) , 2016, 95(8): e2858. DOI: 10.1097/MD.0000000000002858. [28] Costa D, Betancor L, Gadea P, et al. Polyclonal Campylobacter fetus Infections Among Unrelated Patients, Montevideo, Uruguay, 2013-2018[J]. Clin Infect Dis, 2020, 70(6): 1236–1239. DOI: 10.1093/cid/ciz657. [29] Marchand-Senecal X, Bekal S, Pilon P A, et al. Campylobacter fetus Cluster Among Men Who Have Sex With Men, Montreal, Quebec, Canada, 2014-2016[J]. Clin Infect Dis, 2017, 65(10): 1751–1753. DOI: 10.1093/cid/cix610. [30] Hum S, Quinn K, Brunner J, et al. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies[J]. Aust Vet J, 1997, 75(11): 827–831. DOI: 10.1111/j.1751−0813.1997.tb15665.x. [31] 屈平华, 侯水平, 李松, 等. 临床血液标本中18株弯曲菌的菌种与特征分析[J]. 临床检验杂志,2015(02):88–93. DOI: 10.13602/j.cnki.jcls.2015.02.03.Qu PH, Hou SP, Li S, et, al. Type and characterization of 18 strains ofCampylobacter isolated from blood samples[J]. Chin J of Clin Lab Sci, 2015(02): 88–93. DOI: 10.13602/j.cnki.jcls.2015.02.03. [32] Giacomelli M, Piccirillo A. Pet reptiles as potential reservoir of Campylobacter species with zoonotic potential[J]. Vet Rec, 2014, 174(19): 479. DOI: 10.1136/vr.102243. [33] Gilbert M J, Kik M, Timmerman A J, et al. Occurrence, diversity, and host association of intestinal Campylobacter, Arcobacter, and Helicobacter in reptiles[J]. PLoS One, 2014, 9(7): e101599. DOI: 10.1371/journal.pone.0101599. [34] Patrick M E, Gilbert M J, Blaser M J, et al. Human infections with new subspecies of Campylobacter fetus[J]. Emerg Infect Dis, 2013, 19(10): 1678–1680. DOI: 10.3201/eid1910.130883. -