Establishment of Fluorescence qPCR Method for Detection of Staphylococcus Aureus and Its Application in Feces Detection of Rats and Mice

doi:10.12300/j.issn.1674-5817.2023.022

Abstract

Abstract:

Objective To establish a method for rapid and sensitive detection of Staphylococcus aureus. Methods The specific gene nuc of Staphylococcus aureus was selected as the target gene. A pair of specific primers and a TaqMan probe were designed and synthesized according to the published sequence of the nuc gene. Establish a nucleic acid detection method for nuc gene using fluorescence quantitative PCR technology, and apply it clinically in the detection of fecal samples from rats and mice. Results The DNA extracted from Staphylococcus aureus and other non-Staphylococcus aureus strains was detected by qPCR. The results showed that Staphylococcus aureus had a specific amplification curve, while other non-Staphylococcus aureus did not, indicating that the designed primers and probes were specific for Staphylococcus aureus. The sensitivity of this method was determined by diluting the DNA of Staphylococcus aureus by 10 times. The results showed that the detection limit of this method was 10 fg DNA, which was 2 orders of magnitude higher than that of ordinary PCR method. A total of 91 clinical samples were detected in this study, of which 4 rat samples from the same facility had a typical S-curve. The PCR products were sequenced and BLAST compared. The gene sequence of this sample was 100% similar to that of Staphylococcus aureus, indicating that the sample was positive for the nucleic acid of Staphylococcus aureusnuc gene, with a positive rate of 4.40%. The result was consistent with that obtained by bacterial culture method. The nucleic acid extraction adopted a full-automatic nucleic acid purification instrument, and the time required from nucleic acid extraction to detection result determination was less than 1.5 h. Conclusion The qPCR method established in this study to identify Staphylococcus aureus with nuc gene as the target gene has the advantages of fast, high sensitivity and specificity, and can be used for the detection of Staphylococcus aureus in feces of rats and mice.

Key words: Staphylococcus aureus, Method of detection, Fluorescent quantitative PCR, Feces, Rats, Mice

CLC Number:

Q95-33

Lingzhi YU,Jianyun XIE,Liping FENG,et al. Establishment of Fluorescence qPCR Method for Detection of Staphylococcus Aureus and Its Application in Feces Detection of Rats and Mice[J]. Laboratory Animal and Comparative Medicine, 2023, 43(5): 566-573. DOI: 10.12300/j.issn.1674-5817.2023.022.

Figures/Tables 6

References 35

1	AHMAD-MANSOUR N, LOUBET P, POUGET C, et al. Staphylococcus aureus toxins: an update on their pathogenic properties and potential treatments[J]. Toxins, 2021, 13(10):677. DOI: 10.3390/toxins13100677 .
2	HOWDEN B P, GIULIERI S G, WONG FOK LUNG T, et al. Staphylococcus aureus host interactions and adaptation[J]. Nat Rev Microbiol, 2023, 21(6):380-395. DOI: 10.1038/s41579-023-00852-y .
3	TUON F F, SUSS P H, TELLES J P, et al. Antimicrobial treatment of Staphylococcus aureus biofilms[J]. Antibiotics (Basel), 2023, 12(1):87. DOI: 10.3390/antibiotics12010087 .
4	国家市场监督管理总局, 中国国家标准化管理委员会. 实验动物微生物、寄生虫学等级及监测: GB 14922—2022[S]. 北京: 中国标准出版社, 2022.
	State Administration for Market Regulation; Standardization Administration of the People's Republic of China. Laboratory animals—Microbiological and parasitical standards and monitoring: GB 14922-2022[S]. Beijing: Standards Press of China, 2022.
5	中华人民共和国国家质量监督检验检疫总局, 中国国家标准化管理委员会.实验动物微生物学检测方法: GB/T 14926—2001[S]. 北京: 中国标准出版社, 2022.
	General Administration of Quality Supervision, Inspection and Quarantine; Standardization Administration of the People's Republic of China. Laboratory animals—Microbiological examination methods: GB/T 14926-2001[S]. Beijing: Standards Press of China, 2001.
6	翟俊辉, 宋亚军, 杜宗敏, 等. 通用基因芯片检测感染性细菌方法的研究[J]. 中国公共卫生, 2003, 19(4): 430-431. DOI: 10.3321/j.issn: 1001-0580.2003.04.021 .
	ZHAI J H, SONG Y J, DU Z M, et al. Study on universal microarray system for detection of common clinical infectious bacteria[J]. China J Public Health, 2003, 19(4): 430-431.DOI: 10.3321/j.issn: 1001-0580.2003.04.021 .
7	STRAUB J A, HERTEL C, HAMMES W P. A 23S rDNA-targeted polymerase chain reaction-based system for detection of Staphylococcus aureus in meat starter cultures and dairy products[J]. J Food Prot, 1999, 62(10):1150-1156. DOI: 10.4315/0362-028x-62.10.1150 .
8	ONO H K, HIROSE S, NAITO I, et al. The emetic activity of staphylococcal enterotoxins, SEK, SEL, SEM, SEN and SEO in a small emetic animal model, the house musk shrew[J]. Microbiol Immunol, 2017, 61(1):12-16. DOI: 10.1111/1348-0421.12460 .
9	KRONING I S, IGLESIAS M A, MENDONÇA K S, et al. Presence of classical enterotoxin genes, agr typing, antimicrobial resistance, and genetic diversity of Staphylococcus aureus from milk of cows with mastitis in southern Brazil[J]. J Food Prot, 2018, 81(5):738-742. DOI: 10.4315/0362-028X.JFP-17-436 .
10	MATSUDA K, TSUJI H, ASAHARA T, et al. Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR[J]. Appl Environ Microbiol, 2007, 73(1):32-39. DOI: 10.1128/aem.01224-06 .
11	王敏, 范婧, 李先平. PCR扩增法检测耐甲氧西林金黄色葡萄球菌的mecA基因[J]. 广东医学, 2008, 29(4):566-568. DOI: 10.3969/j.issn.1001-9448.2008.04.015 .
	WANG M, FAN J, LI X P. Detection of mecA gene in methicillin-resistant Staphylococcus aureus by PCR amplification[J]. Guangdong Medical Journal, 2008, 29(4):566-568. DOI: 10.3969/j.issn.1001-9448.2008.04.015 .
12	杨波. MecA基因聚合酶链反应扩增法与细菌鉴定仪法对耐甲氧西林金黄色葡萄球菌的鉴定比较及药敏分析[J]. 实用医技杂志, 2017, 24(2):141-142. DOI: 10.19522/j.cnki.1671-5098.2017.02.007 .
	YANG B. Comparison and drug sensitivity analysis of methicillin-resistant Staphylococcus aureus by MecA gene polymerase chain reaction amplification method and bacterial identification instrument method[J]. Journal of Practical Medical Techniques, 2017, 24(2):141-142. DOI: 10.19522/j.cnki.1671-5098.2017.02.007 .
13	FAOUZI, BEKKAOUI, . Rapid detection of the mecA gene in methicillin resistant staphylococci using a colorimetric cycling probe technology[J]. Diagn Microbiol Infect Dis, 1999, 34(2):83-90. DOI: 10.1016/S0732-8893(99)00012-7 .
14	KOBAYASHI N, WU H, KOJIMA K, et al. Detection of mecA, femA, and femB genes in clinical strains of staphylococci using polymerase chain reaction[J]. Epidemiol Infect, 1994, 113(2):259-266. DOI: 10.1017/s0950268800051682 .
15	OU A F, WANG K, MAO Y X, et al. First report on the rapid detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) in viable but non-culturable (VBNC) under food storage conditions[J]. Front Microbiol, 2021, 11:615875. DOI: 10.3389/fmicb.2020.615875 .
16	SHOAIB M, AQIB A I, ALI M M, et al. Tracking infection and genetic divergence of methicillin-resistant Staphylococcus aureus at pets, pet owners, and environment interface[J]. Front Vet Sci, 2022, 9:900480. DOI: 10.3389/fvets.2022.900480 .
17	HENNEKINNE J A, DE BUYSER M L, DRAGACCI S. Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation[J]. FEMS Microbiol Rev, 2012, 36(4):815-836. DOI: 10.1111/j.1574-6976.2011.00311.x .
18	NADIYA S, KOLLA H B, REDDY P N. Optimization and evaluation of a multiplex PCR assay for detection of Staphylococcus aureus and its major virulence genes for assessing food safety[J]. Publ Braz Soc Microbiol, 2023, 54(1):311-321. DOI: 10.1007/s42770-023-00906-6 .
19	GONET M, KROWARSCH D, SCHUBERT J, et al. Stability and resistance to proteolysis of enterotoxins SEC and SEL produced by Staphylococcus epidermidis and Staphylococcus aureus [J]. Foodborne Pathog Dis, 2023, 20(1):32-37. DOI: 10.1089/fpd.2022.0059 .
20	MORADIKIAN M, KOMIJANI M, SHAYESTEHFAR A. Detection of Staphylococcus aureus and their toxin genes inhabit on the scorpions surface[J]. Arch Microbiol, 2022, 204(9):576. DOI: 10.1007/s00203-022-03176-2 .
21	SU Y C, WONG A C. Identification and purification of a new staphylococcal enterotoxin, H[J]. Appl Environ Microbiol, 1995, 61(4):1438-1443. DOI: 10.1128/aem.61.4.1438-1443.1995 .
22	MUNSON S H, TREMAINE M T, BETLEY M J, et al. Identification and characterization of staphylococcal enterotoxin types G and I from Staphylococcus aureus [J]. Infect Immun, 1998, 66(7):3337-3348. DOI: 10.1128/IAI.66. 7.3337-3348.1998 .
23	GERGOVA R, TSITOU V M, DIMOV S G, et al. Molecular epidemiology, virulence and antimicrobial resistance of Bulgarian methicillin resistant Staphylococcus aureus isolates[J]. AMicr, 2022, 69(3):193-200. DOI: 10.1556/030.2022. 01766 .
24	TUCKER P W, HAZEN E E Jr, COTTON F A. Staphylococcal nuclease reviewed: a prototypic study in contemporary enzymology. I. Isolation; physical and enzymatic properties[J]. Mol Cell Biochem, 1978, 22(2-3):67-77. DOI: 10.1007/BF00496235 .
25	BRAKSTAD O G, MÆLAND J A. Generation and characterization of monoclonal antibodies against Staphylococcus aureus thermonuclease[J]. APMIS, 1989, 97(1-6):166-174. DOI: 10.1111/j.1699-0463.1989.tb00772.x .
26	KOVACEVIC S, VEAL L E, HSIUNG H M, et al. Secretion of staphylococcal nuclease by Bacillus subtilis [J]. J Bacteriol, 1985, 162(2):521-528. DOI: 10.1128/jb.162.2.521-528.1985 .
27	CHESNEAU O, ALLIGNET J, SOLH N EL. Thermonuclease gene as a target nucleotide sequence for specific recognition of Staphylococcus aureus [J]. Mol Cell Probes, 1993, 7(4):301-310. DOI: 10.1006/mcpr.1993.1044 .
28	MADISON B M, BASELSKI V S. Rapid identification of Staphylococcus aureus in blood cultures by thermonuclease testing[J]. J Clin Microbiol, 1983, 18(3):722-724. DOI: 10.1128/jcm.18.3.722-724.1983 .
29	CANNING B, MOHAMED I, WICKRAMASINGHE N, et al. Thermonuclease test accuracy is preserved in methicillin-resistant Staphylococcus aureus isolates[J]. J Med Microbiol, 2020, 69(4):548-551. DOI: 10.1099/jmm.0.001166 .
30	王纯, 张若鸿, 王晓芳, 等. 乳制品中金黄色葡萄球菌PCR快速检测方法的建立[J]. 核农学报, 2022, 36(6): 1193-1203. DOI: 10.11869/j.issn.100-8551.2022.06.1193 .
	WANG C, ZHANG R H, WANG X F, et al. Development of PCR rapid detection method for Staphylococcus aureus in dairy products[J]. J Nucl Agric Sci, 2022, 36(6): 1193-1203. DOI: 10.11869/j.issn.100-8551.2022.06.1193 .
31	DONG Q S, WANG Q, ZHANG Y, et al. Prevalence, antimicrobial resistance, and staphylococcal toxin genes of bla_TEM-1a-producing Staphylococcus aureus isolated from animals in Chongqing, China[J]. Vet Med Sci, 2023, 9(1):513-522. DOI: 10.1002/vms3.1028 .
32	BRAKSTAD O G, AASBAKK K, MAELAND J A. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene[J]. J Clin Microbiol, 1992, 30(7):1654-1660. DOI: 10.1128/jcm.30.7.1654-1660.1992 .
33	高正琴, 邢华, 李厚达. PCR技术在鼠金黄色葡萄球菌检测中的初步研究[J]. 畜牧与兽医, 2002, 34(8):5-7. DOI: 10.3969/j.issn.0529-5130.2002.08.003 .
	GAO Z Q, XING H, LI H D. The pilot study of polymerase chain reaction for detecting Staphylococcus aureus in rats and mice[J]. Animal Husb Vet Med, 2002, 34(8):5-7. DOI: 10.3969/j.issn.0529-5130.2002.08.003 .
34	王鹏飞. 实验动物金黄色葡萄球菌、绿脓杆菌和肺支原体多重PCR检测方法的建立与初步应用[D]. 石家庄:河北医科大学,2016.
	WANG P F. The Establishment and preliminary applications of Multiple PCR for detecting Staphylococcus aureus,Pseudomonas aeruginosa and Mycoplasma pulmonis in Laboratory Animals[D]. SHIJIAZHUANG: Hebei Medical University, 2016.
35	陆文俊. 金黄色葡萄球菌特异性靶点的筛选与PCR快速检测方法的建立[D]. 南京:南京农业大学, 2017.
	LU W J. The screening of specific target for Staphylococcus aureus and establishment of rapid PCR detection method[D]. Nanjing: Nanjing Agriculture University. 2017.

DNA量 DNA quantities	5次检测结果（Ct值） 5 test results(Ct value)					DNA量 DNA quantities	5次检测结果（Ct值） 5 test results(Ct value)
DNA量 DNA quantities	1	2	3	4	5	DNA量 DNA quantities	1	2	3	4	5
10 pg	33.13	34.08	36.00	34.19	33.02	10 fg	26.04	26.10	26.20	26.12	26.08
	34.44	32.27	33.47	34.04	34.56		25.80	25.89	25.83	26.15	26.10
	32.72	32.60	32.35	31.90	32.56		26.17	25.39	25.86	25.76	25.91
	32.13	32.88	34.21	33.14	32.90		25.87	25.96	25.86	25.90	25.66
	33.83	35.05	32.60	33.99	33.41		25.86	25.78	26.10	26.12	25.69
平均数1 Mean 1	33.25	33.38	33.73	33.45	33.29		25.95	25.82	25.97	26.01	25.89
平均数2 Mean 2	33.42						25.93
标准差SD	0.19						0.19
CV/%	0.56						0.75

DNA量 DNA quantities	5次检测结果（Ct值） 5 test results(Ct value)					DNA量 DNA quantities	5次检测结果（Ct值） 5 test results(Ct value)
DNA量 DNA quantities	1	2	3	4	5	DNA量 DNA quantities	1	2	3	4	5
10 pg	33.13	34.08	36.00	34.19	33.02	10 fg	26.04	26.10	26.20	26.12	26.08
	34.44	32.27	33.47	34.04	34.56		25.80	25.89	25.83	26.15	26.10
	32.72	32.60	32.35	31.90	32.56		26.17	25.39	25.86	25.76	25.91
	32.13	32.88	34.21	33.14	32.90		25.87	25.96	25.86	25.90	25.66
	33.83	35.05	32.60	33.99	33.41		25.86	25.78	26.10	26.12	25.69
平均数1 Mean 1	33.25	33.38	33.73	33.45	33.29		25.95	25.82	25.97	26.01	25.89
平均数2 Mean 2	33.42						25.93
标准差SD	0.19						0.19
CV/%	0.56						0.75

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