Specific molecular sequence and method for rapidly detecting enterococcus hila by using specific molecular sequence
By designing a method combining specific molecular sequences and PCR, the problems of long detection time, high cost and false negatives in the prior art are solved, and a fast, accurate and low-cost detection effect is achieved.
Patent Information
- Application Number
- CN202311256954.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-27
- Publication Date
- 2025-03-28
AI Technical Summary
The existing Enterococcus Hilac test method has the defects of long detection time, high cost, cumbersome operation and false positive false negative results, which is difficult to meet the needs of fast, accurate and low-cost testing.
A class of specific molecular sequences was designed, and the specific amplification primer pair was combined with PCR method to rapidly amplify and detect the amplification product by gel electrophoresis to achieve rapid detection of Enterococcus Hilac.
This method can complete the detection in a short time, reduces costs, is simple to operate, has strong specificity, avoids false positive or false negative results, and is highly practical.
Smart Images
Figure CN119709729A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microbial testing, and in particular relates to a class of specific molecular sequences and a method for rapidly detecting Enterococcus hilaris using the same. Background Art
[0002] Enterococcus hirae belongs to the genus Enterococcus (Enterococcus spp.), which is a Gram-positive coccus with a round or oval shape and is distributed alone or in short chains. It is a normal flora in the intestines of humans and animals. As a normal flora in the intestines of humans and animals, enterococci generally do not cause diseases, but when the body's immunity decreases, it will become a conditional pathogen and cause a variety of infections, such as urinary tract infections, wound infections, meningitis, endocarditis, etc., which can be life-threatening in severe cases (Maekawa, T.; Fukaya, R.; Takamatsu, S.; Itoyama, S.; Miyoshi, E., Possible involvement of Enterococcus infection in the pathogenesis of chronic pancreatitis and cancer. Biochemical and Biophysical Research Communications 2018, 506, 962-969). Studies have shown that during the period of 2011-2014, enterococci were the second most common pathogen causing hospital-acquired infections, accounting for 14% of the total number of cases. Their detection rate was second only to Escherichia coli, and exceeded that of Pseudomonas aeruginosa and Staphylococcus aureus (Lindsey MW; Amy KW; Brandi L.; Margaret AD; Jean P.; Alexander JK; Jonathan RE; Dawn MS, Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the national healthcare safety network at the centers for disease control and prevention, 2011-2014. Infect Control Hosp Epidemiol 2016, 37, 1288-1301.). The wide distribution and strong environmental adaptability of enterococci may be important reasons for their ability to cause clinical infections.
[0003] The detection methods of Enterococcus hilaris mainly include traditional physiological and biochemical reactions, API biochemical identification strips, and microbial automatic identification instruments. These methods have different advantages and disadvantages, such as: the results of traditional physiological and biochemical reactions are relatively accurate, but the operation steps are relatively cumbersome, and sometimes additional tests are required for identification, which is time-consuming; API biochemical identification strips have the advantages of simple trace detection technology and can identify almost all bacteria, but their disadvantages are that the systems vary greatly, the price is relatively expensive, and there are many false positives in the test results; microbial automatic identification instruments are relatively easy to operate, but the price is relatively expensive. Molecular biological detection methods based on PCR have gradually become one of the most promising detection technologies to replace traditional detection methods due to their rapid, accurate, and simple characteristics, and the specificity of target sequences is the key to PCR detection technology. Therefore, systematically exploring new specific targets and establishing accurate and rapid PCR detection methods are of great significance for the clinical detection of Enterococcus hilaris and the investigation and research on the epidemic transmission law. Summary of the invention
[0004] The purpose of the present invention is to provide a class of specific molecular sequences and a method for quickly detecting Enterococcus hilarius, which has the advantages of short detection time, low detection cost, simple operation, strong specificity, etc. Enterococcus hilarius can be detected without biochemical identification, and is more practical.
[0005] To achieve the above object, the present invention adopts the following technical solutions:
[0006] A type of specific molecular sequence, the nucleotide base sequence of the specific molecular sequence is shown as SEQ ID No.1-SEQ ID No.26.
[0007] The present invention also provides a method for rapidly detecting Enterococcus silencingii using the above-mentioned specific molecular sequence, and the specific method steps are as follows:
[0008] (1) designing a specific amplification primer pair according to any specific molecular sequence in the base sequences SEQ ID NO.1-SEQ ID NO.26;
[0009] (2) Extract sample DNA and amplify using PCR;
[0010] (3) Detect the amplified product by gel electrophoresis. If a corresponding single amplified band appears in the electrophoresis result, it is judged that the sample contains the target bacteria. Otherwise, it is judged that the sample does not contain the target bacteria.
[0011] Preferably, in step (1), the base sequences SEQ ID NO.1-SEQ ID NO.26 correspond to the base sequences of the designed specific amplification primer pairs as shown in SEQ ID NO.1F-SEQ ID NO.1R, SEQ ID NO.2F-SEQ ID NO.2R, ..., SEQ ID NO.25F-SEQ ID NO.25R, and SEQ ID NO.26F-SEQ ID NO.26R.
[0012] Preferably, in step (2), the PCR method selects any specific amplification primer pair to form a PCR detection system, and the specific amplification parameters are:
[0013] The PCR detection system is 25 μL, including: 2×PCRMix 12.5 μL, template DNA 1 μL, 1.0 μL of 10 μM upstream and downstream primers, and sterile double distilled water to make up the volume to 25 μL;
[0014] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 50-62°C for 30 s; extension at 72°C for 30-90 s, for a total of 35 cycles; and extension at 72°C for 10 min.
[0015] Compared with the prior art, the detection method of the present invention has the advantages of short detection time and can detect Enterococcus hilarius without biochemical identification, thus reducing the detection cost; at the same time, the detection method of the present invention can be used to detect certain strains with unclear interspecies differences, thus avoiding false positive or false negative results, and being more practical; the specific molecular sequence of the present invention, i.e., 26 conserved nucleic acids, has a single specificity, and the detection is accurate and the result determination is simple. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 The electrophoresis diagram of the nucleotide base sequences SEQ ID NO.1-SEQ ID NO.26 of Enterococcus hilaris in Example 2;
[0017] Figure 2 The electrophoresis results of PCR detection of the specific molecular sequence of Enterococcus hilaris in Example 2 are shown in FIG. 1 , FIG. 2 , FIG. 3 , FIG. 4 , FIG. 5 , FIG. 6 , FIG. 7 , FIG. 8 , FIG. 9 , FIG. 10 , FIG. 11 , FIG. 12 , FIG. 13 , FIG. 14 , FIG. 15 , FIG
[0018] Figure 3 This is a graph showing the electrophoresis results for the specificity evaluation of the PCR detection method for Enterococcus silencing in Example 2; in the graph, Figures (a)-(g) are SEQ ID NO.1-SEQ ID NO.26, respectively. DETAILED DESCRIPTION
[0019] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.
[0020] The present invention investigated the distribution of microorganisms in 1,300 human samples from Jiaoling County, my country's "World Longevity Village", and isolated and preserved 228 strains of enterococci, of which 53.51% were Enterococcus faecalis, 13.16% were Enterococcus avium, 6.58% were Enterococcus faecium, 4.39% were Enterococcus serratus, 3.51% were Enterococcus casei, and 18.86% were other Enterococci, providing a strong guarantee for the mining and verification of specific targets of the present invention. Therefore, the human isolates involved in the following embodiments are all microbial isolates obtained from human samples in Jiaoling County, Guangdong Province, my country.
[0021] This embodiment 1
[0022] This embodiment provides a type of specific molecular sequence, the nucleotide base sequence of the specific molecular sequence is shown as SEQ ID No.1-SEQ ID No.26.
[0023] This embodiment 2
[0024] This embodiment provides a method for using the above-mentioned specific molecular sequence to quickly detect Enterococcus hilaris, and the specific method steps are as follows:
[0025] (1) Primer design: A specific amplification primer pair is designed according to any specific molecular sequence in the base sequence SEQ ID NO.1-SEQ ID NO.26. The primer pair sequence is shown in Table 1 below:
[0026] Table 1 Primer sequence pairs for specific PCR detection of Enterococcus serrata
[0027]
[0028]
[0029]
[0030] (2) DNA template preparation: The Enterococcus hilaris strain was cultured in LB liquid medium, and the bacterial genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit as a test template; PCR amplification was performed, and the PCR detection system and amplification procedure were as follows:
[0031] The PCR detection system is 25 μL, including: 2×PCR Mix 12.5 μL, template DNA 1 μL, 1.0 μL of 10 μM upstream and downstream primers, and sterile double distilled water to make up the volume to 25 μL;
[0032] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 90 s, for a total of 35 cycles; and extension at 72°C for 10 min.
[0033] (3) Determination of PCR test results:
[0034] The amplified products were detected by gel electrophoresis. The electrophoresis results were observed to see whether there were single amplified bands at the positions of 390, 936, 1621, 707, 311, 548, 216, 426, 767, 349, 383, 147, 288, 231, 166, 317, 463, 417, 263, 153, 284, 164, 284, 191, 146, and 450 bp, respectively. If so, it indicated that the sample contained Enterococcus hirae. If no corresponding single amplified band appeared, the sample did not contain Enterococcus hirae.
[0035] Results of specificity evaluation of PCR detection method
[0036] 8 strains of Enterococcus hilaris, 39 strains of other enterococci including Enterococcus casei, Enterococcus faecalis, Enterococcus faecium, and 16 strains of non-enterococci including Escherichia coli and Shigella were tested by PCR according to the above method. The results are as follows Figure 1-3 As shown, in the detection of 26 primer sequence pairs, Enterococcus hirae showed specific amplification bands. Except for some Enterococcus casei, other enterococci and non-enterococcal strains had no specific bands. The strains used and the test results are shown in Table 2 below. In the test result column in the table, "+" indicates positive and "-" indicates negative. Table 2 shows that only the test result of Enterococcus hirae is positive, which shows that the detection method obtained in this embodiment has a strong specificity for the detection of Enterococcus hirae.
[0037] Table 2 Results of the specific evaluation test of Enterococcus hilaris in Example 2
[0038]
[0039]
[0040] Example 3
[0041] Detection of suspected strains of Enterococcus shiratus
[0042] The PCR detection method of Enterococcus hirae in Example 2 was used to detect 73 suspected strains of Enterococcus isolated from human samples. The human samples were collected from the feces and urine of centenarians in Guangfu Town, Jiaoling County, Guangdong Province. The sample processing and isolation of suspected strains refer to the national standard method (GB / T 38735-2020). Using the method of the present invention, the number of strains of Enterococcus hirae was 6, and other suspected strains were identified as other species of Enterococcus (Table 3), which is consistent with the 16s rDNA identification results. This example illustrates that the 26 PCR detection methods of Enterococcus hirae of the present invention are highly reliable.
[0043] Table 3 Detection of suspected strains of Enterococcus
[0044] Species name Detection method of the present invention 16s rDNA identification E.casseliflavus 0 3 E.faecalis 0 34 E. faecium 0 4 E.hirae 6 6 E.avium 0 10 E.galinarum 0 4 E. raffinosus 0 2 E.thailandicus 0 1 E.lactis 0 1 Enterococcus spp. 0 8
Claims
1. A class of specific molecular sequences, characterized in that: The nucleotide base sequences of the specific molecular sequences are shown as SEQ ID No.1-SEQ ID No.
26.
2. The method for rapid detection of Enterococcus silencingii using a specific molecular sequence according to claim 1, characterized in that: The specific steps are as follows: (1) designing a specific amplification primer pair according to any specific molecular sequence in the base sequences SEQ ID NO.1-SEQ ID NO.26; (2) Extract sample DNA and amplify using PCR; (3) Detect the amplified product by gel electrophoresis. If a corresponding single amplified band appears in the electrophoresis result, it is judged that the sample contains the target bacteria. Otherwise, it is judged that the sample does not contain the target bacteria.
3. The method for rapid detection of Enterococcus silencingii using a specific molecular sequence according to claim 2, characterized in that: In step (1), the base sequences SEQ ID NO.1-SEQ ID NO.26 correspond to the base sequences of the designed specific amplification primer pairs as shown in SEQ ID NO.1F-SEQ ID NO.1R, SEQ ID NO.2F-SEQ ID NO.2R, ..., SEQ ID NO.25F-SEQ ID NO.25R, and SEQ ID NO.26F-SEQ ID NO.26R.
4. The method for rapid detection of Enterococcus silencingii using a specific molecular sequence according to claim 2 or 3, characterized in that: In step (2), the PCR method selects any specific amplification primer pair to form a PCR detection system, and the specific amplification parameters are: The PCR detection system is 25 μL, including: 2×PCR Mix 12.5 μL, template DNA 1 μL, 1.0 μL of 10 μM upstream and downstream primers, and sterile double distilled water to make up the volume to 25 μL; The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 50-62°C for 30 s; extension at 72°C for 30-90 s, for a total of 35 cycles; and extension at 72°C for 10 min.