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, Xiaofeng WEI. 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.

Figures/Tables 6

Figure 1 Specificity for Staphylococcus aureus detected by real-time fluorescent quantitative PCRNote：Red S-type curve represents the positive control.RFU， Relative fluorescence unit.

Figure 2 Standard curve （A） and sensitivity （B） for Staphylococcus aureus detected by real-time fluorescent quantitative PCRNote：The amounts of nucleic acid represented by the curves from left to right is 1 ng， 100 pg， 10 pg， 1 pg， 100 fg， 10 fg and 1 fg， respectively.

Table 1 Intragroup repeatability data (Ct) and coefficient of variation (CV)

DNA量

DNA quantities

10次检测结果（Ct值）

10 test results (Ct value)

CV值/%

CV value/%

Table 2 Intergroup repeatability data (Ct) and coefficient of variation（CV）

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

Figure 3 Clinical detection results for Staphylococcus aureus by real-time fluorescent quantitative PCR detectionNote：The red S curve represents the positive control for amplification， the black S curve represents the positive control for nucleic acid extraction， and the four blue S curves represent the positive samples.

Figure 4 Sequence of PCR product （A） and the result of BLAST （B）

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.

[1]	Jinhua HU, Jingjie HAN, Min JIN, Bin HU, Yuefen LOU. Effects of Puerarin on Bone Density in Rats and Mice: A Meta-analysis [J]. Laboratory Animal and Comparative Medicine, 2024, 44(2): 149-161.
[2]	Min LIANG, Yang GUO, Jinjin WANG, Mengyan ZHU, Jun CHI, Yanjuan CHEN, Chengji WANG, Zhilan YU, Ruling SHEN. Construction of Dmd Gene Mutant Mice and Phenotype Verification in Muscle and Immune Systems [J]. Laboratory Animal and Comparative Medicine, 2024, 44(1): 42-51.
[3]	Jianhua ZHENG, Yunzhi FA, Qiaoyan DONG, Yefeng QIU, Jingqing CHEN. Construction and Evaluation of a Mouse Model with Intestinal Injury by Acute Hypoxic Stress in Plateau [J]. Laboratory Animal and Comparative Medicine, 2024, 44(1): 31-41.
[4]	Qianqian TANG, Xiuli ZHANG, Zai CHANG. Statistical Analysis of the Leakage Situation in the Automated Watering System for Mice in Tsinghua University Laboratory Animal Resources Center [J]. Laboratory Animal and Comparative Medicine, 2024, 44(1): 85-91.
[5]	Xin LIU, Shaobo SHI, Cui ZHANG, Bo YANG, Chuan QU. Construction and Evaluation of End-to-side Anastomosis Model of Autologous Arteriovenous Fistula in Mice [J]. Laboratory Animal and Comparative Medicine, 2023, 43(6): 595-603.
[6]	Dan WANG, Xiaolu ZHANG, Yan WANG, Bo FU, Wendong WANG, Jing LIU, Suyin ZHANG, Yihe WU, Deguo WU, Xiaoyan DU, Dawei ZHAN, Xiulin ZHANG, Changlong LI. Study on the Antibody Production Efficiency in Modified Big-BALB/c Mice [J]. Laboratory Animal and Comparative Medicine, 2023, 43(6): 612-618.
[7]	Liya ZHAO, Liju NI, Caiqin ZHANG, Jianping TANG, Yangzheng YAO, Yanyan NIE, Xiaoxue GU, Ying ZHAO. Establishing a Genetic Detection Protocol of Single Nucleotide Polymorphisms Panels in Inbred Rats Based on Multiplex PCR-LDR [J]. Laboratory Animal and Comparative Medicine, 2023, 43(5): 548-558.
[8]	Chengji WANG, Jue WANG, Haijie WANG, Weisheng LU, Yan SHI, Zhengye GU, Mingqiu WAN, Ruling SHEN. Application of Optimized Latex Perfusion Technique in the Establishment of Craniofacial Venous Model in Mice [J]. Laboratory Animal and Comparative Medicine, 2023, 43(5): 574-578.
[9]	Shanshan ZHAI, Liang LIANG, Yingying CAO, Zhuxin LI, Qing WANG, Junyu TAO, Chenxia YUN, Jing LENG, Haibo TANG. Diagnosis of Trichoepithelioma in a Tree Shrew and Observation of Cell Biological Characteristics [J]. Laboratory Animal and Comparative Medicine, 2023, 43(4): 440-445.
[10]	Xiaoqian TAN, Hao YANG, Huiqing TANG, Wei QU, Liang LI, Zhen QIAN, Jianzhong GU, Ping XU, Junhua XIAO. Creation and Analysis of Related Genetic Characteristics of BALB/cA.Cg.SHJH ^hr Mice [J]. Laboratory Animal and Comparative Medicine, 2023, 43(4): 363-370.
[11]	Yasheng DENG, Jiang LIN, Chiling GAN, Guanfeng ZENG, Jiayin HUANG, Huifang DENG, Yingxian MA, Siyin HAN. Literature Analysis of the Preparation Elements of Animal Models of Skin Photoaging and the Data of Subjects [J]. Laboratory Animal and Comparative Medicine, 2023, 43(4): 406-414.
[12]	Zhiqiang PAN, Zixin NONG, Haina XIE, Peike PENG. Injurious Effect of Cisplatin on the Function of Hypothalamus-pituitary-adrenal/gonadal Axis in Mice and the Intervention Effect of Dehydroepiandrosterone [J]. Laboratory Animal and Comparative Medicine, 2023, 43(3): 229-242.
[13]	Wenwen GUO, Ya ZHAO, Yinghua WANG, Ke LIU, Xu GE, Yanying ZHANG, Yongfeng WANG, Changhong SHI. Repairing Effects of Ginsenoside Rg1 on Traumatic Brain Injury in Mice [J]. Laboratory Animal and Comparative Medicine, 2023, 43(3): 243-252.
[14]	Yuan ZHANG, Han LI, Chengfang ZHANG. Whole-brain Transcriptomic Analysis of Weight Gain Mice induced by Olanzapine [J]. Laboratory Animal and Comparative Medicine, 2023, 43(3): 262-270.
[15]	Zhigang TAN, Jinxin LIU, Chuya ZHENG, Wenfeng LIAO, Luping FENG, Hongli PENG, Xiu YAN, Zhenjian ZHUO. Advances and Applications in Animal Models of Neuroblastoma [J]. Laboratory Animal and Comparative Medicine, 2023, 43(3): 288-296.