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）

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