Specific molecular target, kit and detection method for simultaneously detecting important pathogenic species in enterococcus

Abstract

The invention discloses specific molecular targets for simultaneously detecting important pathogenic species in enterococcus, a kit and a detection method thereof, the specific molecular targets are respectively an enterococcus specific identification target rmC and three enterococcus species specific targets ECBG02351, EGM181RS15835 and DQL78RS08525, then specific primer pairs are respectively designed for the four specific molecular targets, and the specific primer pairs are respectively used for detecting the important pathogenic species in the enterococcus. A multiplex PCR detection system is constructed, whether a sample containing multiple pathogenic species genome DNA contains enterococcus strains or not can be rapidly and accurately detected and identified, whether the enterococcus strains are enterococcus pluvialis, quail chicken enterococcus and cecum enterococcus or not can be determined, the detection cost is reduced, and the detection efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and specifically relates to a specific molecular target, a kit and a detection method for simultaneously detecting important pathogenic species within the genus Enterococcus. Background Art

[0002] The genus Enterococcus (Enterococcus Thiercelin and Jouhaud, 1984) is a Gram-positive coccus, with a round or oval shape, distributed singly or in short chains. It is a normal inhabitant of the intestines of humans and animals and shows intrinsic resistance to many antibacterial drugs, such as compound sulfamethoxazole, cephalosporins, clindamycin, and low concentrations of aminoglycosides. Currently, Enterococcus is an important nosocomial infection pathogen among Gram-positive bacteria, second only to the genus Staphylococcus. The most common infections are urinary tract infections, followed by infections in areas such as the abdomen and pelvis after trauma and surgery. The most frequently isolated species clinically is Enterococcus faecalis, followed by Enterococcus faecium. In the case of a history of bird contact, the infection probability of Enterococcus avium is also relatively high. Therefore, it is very necessary to detect pathogenic species within the genus Enterococcus.

[0003] Chinese Patent CN 101880728 B discloses a multiplex PCR detection primer for the genus Enterococcus, including a primer for the genus Enterococcus, a primer for Enterococcus faecalis, and a primer for Enterococcus faecium. It can identify the genus Enterococcus and Enterococcus faecalis or Enterococcus faecium in one PCR, can detect at least 1 ng of genomic DNA, has high sensitivity and relatively strong specificity, which is beneficial to the identification and diagnosis of Enterococcus. However, its screening was carried out around 2010, and the screening process only included the genomes of a few species. Therefore, the gene specificity of the screened genus Enterococcus needs to be improved.

[0004] Chinese Patent CN 116287329 A discloses specific molecular targets for identifying four species within the genus Enterococcus and a rapid detection method thereof, including specific molecular targets for identifying Enterococcus casseliflavus, Enterococcus faecalis, Enterococcus faecium, and Enterococcus hellenicus, and primer sets are designed for these four specific molecular targets. The species are identified by PCR amplification and recognition of the amplification products. However, the specific molecular targets obtained by its screening only include 4 species and do not include genus-specific genes. Therefore, it can only specifically detect any one or more of these 4 Enterococcus faecalis species. When these four Enterococcus species are not present, it cannot help to determine whether other species within the genus Enterococcus are present, and the accuracy of the detection result is only guaranteed at the species level.

[0005] Due to the diversity and complexity of the genomes of microorganisms, they can undergo significant genetic changes in a short period of time through mechanisms such as mutation, selection, and genetic drift to adapt to environmental changes. Moreover, horizontal gene transfer frequently occurs among microorganisms, leading to frequent changes in the genomes of microorganisms. Therefore, the specific molecular targets used for microorganism detection and identification need to have broad applicability to improve the reliability and repeatability of experiments. Therefore, for the detection and identification of important pathogenic species within the genus Enterococcus, it is necessary to select conserved genes as specific molecular targets, and it is necessary to first clarify whether the sample contains bacteria of the genus Enterococcus before further detecting and identifying whether it is an important pathogenic species. Therefore, it is necessary to screen and obtain conserved genes that only exist in the genus Enterococcus and conserved genes that only exist in its important pathogenic species as specific molecular targets, and design specific primer pairs to assist in the detection and identification of important pathogenic species within the genus Enterococcus. Summary of the Invention

[0006] In view of the above technical problems, the present invention provides a specific molecular target, a kit, and a detection method for simultaneously detecting important pathogenic species within the genus Enterococcus. Four specific molecular targets have been screened out, and primer pairs have been designed to construct a multiplex PCR detection system, which can quickly, accurately, and efficiently detect and identify three pathogenic species within the genus Enterococcus: Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus cecorum.

[0007] To achieve the above object, the present invention provides a specific molecular target for simultaneously detecting important pathogenic species within the genus Enterococcus, and the specific molecular target includes a specific identification target for the genus Enterococcus rpmC and three species-specific targets for Enterococcus ECBG_02351 , EGM181_RS15835 , DQL78_RS08525 or one or more of them.

[0008] Preferably, the ECBG_02351 is a specific target for Enterococcus casseliflavus, and its nucleotide sequence is SEQ ID NO:2 and its homologous sequences.

[0009] Preferably, the EGM181_RS15835 is a specific target for Enterococcus gallinarum, and its nucleotide sequence is SEQ ID NO:3 and its homologous sequences.

[0010] Preferably, the DQL78_RS08525 is a specific target for Enterococcus cecorum, and its nucleotide sequence is SEQ ID NO:4 and its homologous sequences.

[0011] Preferably, the nucleotide sequence of the rpmC is SEQ ID NO:1 and its homologous sequences.

[0012] Further preferably, the homologous sequence refers to a sequence identity of 85% or more.

[0013] The present invention also provides a kit for simultaneously detecting important pathogenic species within the genus Enterococcus, which kit comprises a specific primer pair composition, a PCR reaction solution, a positive control, and a negative control.

[0014] Preferably, the specific primer pair composition comprises primer pairs for specific identification targets of the genus Enterococcus rpmC , primer pairs for specific targets of Enterococcus casseliflavus ECBG_02351 , primer pairs for specific targets of Enterococcus gallinarum EGM181_RS15835 , and primer pairs for specific targets of Enterococcus cecorum DQL78_RS08525 , and two or more pairs of them.

[0015] Further preferably, the sequences of the primer pairs for rpmC are SEQ ID NO: 5 and SEQ ID NO: 6 respectively; the sequences of the primer pairs for ECBG_02351 are SEQ ID NO: 7 and SEQ ID NO: 8 respectively; EGM181_RS15835 the sequences of the primer pairs for DQL78_RS08525 are SEQ ID NO: 9 and SEQ ID NO: 10 respectively;

[0016] The present invention also provides a detection method for the kit for simultaneously detecting important pathogenic species within the genus Enterococcus, comprising the following steps: (1) Extracting the DNA of the sample to be tested; (2) Using the extracted DNA as a template, establishing a duplex PCR reaction system with the specific primer pair composition and the PCR reaction solution, and performing a PCR reaction; (3) Determining the reaction result: If specific fragments with fragment sizes of 161 bp, 285 bp, 629 bp, and 417 bp are amplified, the sample to be tested contains Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus cecorum; If specific fragments with fragment sizes of 161 bp, 285 bp, and 629 bp are amplified, the sample to be tested contains Enterococcus casseliflavus and Enterococcus gallinarum; If specific fragments with fragment sizes of 161 bp, 285 bp, and 417 bp are amplified, the sample to be tested contains Enterococcus casseliflavus and Enterococcus cecorum; If specific fragments with fragment sizes of 161 bp, 629 bp, and 417 bp are amplified, the sample to be tested contains Enterococcus gallinarum and Enterococcus cecorum; Specific fragments with sizes of 161bp and 285bp are amplified, indicating that Enterococcus casseliflavus is present in the sample to be tested; Specific fragments with sizes of 161bp and 629bp are amplified, indicating that Enterococcus gallinarum is present in the sample to be tested; Specific fragments with sizes of 161bp and 417bp are amplified, indicating that Enterococcus caecorum is present in the sample to be tested; A specific fragment with a size of 161bp is amplified, indicating that Enterococcus spp. is present in the sample to be tested, but Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus caecorum are absent; No specific fragment is amplified, indicating that none of the pathogenic bacteria of Enterococcus spp. is present in the sample to be tested.

[0017] The beneficial effects of the present invention are as follows: Through large genomics analysis and sequence alignment, a specific molecular target of Enterococcus spp. is screened from 35,104 Enterococcus genomes and 1,426,688 non-Enterococcus genomes (common environmental microorganisms and human pathogenic bacteria belonging to 49 genera). rpmC A specific molecular target of Enterococcus casseliflavus ECBG_ 02351 A specific molecular target of Enterococcus gallinarum EGM181_RS15835 A specific molecular target of Enterococcus caecorum DQL78_RS08525 ; Each specific molecular target not only has high specificity but also high conservation, and can maintain high stability during processes such as genomic mutation, selection, and genetic drift of Enterococcus. Therefore, detection and identification can be carried out with high accuracy, improving the sensitivity and specificity of detection, and the detection work has high repeatability.

[0018] For the obtained 4 specific molecular targets, specific primer pairs are designed respectively. The primer pairs are mixed as a primer composition, and it can be detected by a single PCR whether the sample contains these 3 pathogenic species, and by the size of the amplified fragment, it can be directly determined which pathogenic species it is. Even if Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus caecorum are not present in the sample template, it can also detect and identify whether there are strains within Enterococcus spp. in the sample, helping to further narrow the detection range.

[0019] By combining the specific molecular target of the genus with the specific molecular target of the species, the detection accuracy can be further improved, avoiding false positives and interference from other strains. Only when both the specific molecular target band of the genus and the specific molecular target band of the species appear simultaneously, can it be accurately judged that the pathogenic species in Enterococcus spp. is present, improving the detection accuracy and avoiding false positives. Description of the Drawings

[0020] Figure 1For the conserved single-copy core genes of Enterococcus in Example 1, in the figure, A is the LS-BSR analysis diagram of each conserved gene between Enterococcus and non-Enterococcus; B is the BSR value distribution of Enterococcus-specific genes rpmC

[0021] Figure 2 It is the LS-BSR analysis result diagram of the conserved single-copy core genes of Enterococcus casseliflavus and other species of Enterococcus in Example 2.

[0022] Figure 3 For the specific genes in Example 2 ECBG_02351 It is the diagram of the BSR value distribution of ECBG_02351 in Enterococcus casseliflavus, other species of Enterococcus, and other bacteria.

[0023] Figure 4 It is the LS-BSR analysis result diagram of the conserved single-copy core genes of Enterococcus gallinarum and other species of Enterococcus in Example 3.

[0024] Figure 5 For the specific genes in Example 3 EGM181_RS15835 It is the diagram of the BSR value distribution of EGM181_RS15835 in Enterococcus gallinarum, other species of Enterococcus, and other bacteria.

[0025] Figure 6 It is the LS-BSR analysis result diagram of the conserved single-copy core genes of Enterococcus caecimuris and other species of Enterococcus in Example 4.

[0026] Figure 7 For the specific genes in Example 4 DQL78_RS08525 It is the diagram of the BSR value distribution of DQL78_RS08525 in Enterococcus caecimuris, other species of Enterococcus, and other bacteria.

[0027] Figure 8 For the Enterococcus-specific gene in Example 5 rpmC It is the single-PCR gel electrophoresis diagram of the primer pair of rpmC . In the figure, M is the DNA Marker. Lanes 1-6 are respectively using Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus casseliflavus, Enterococcus gallinarum, Enterococcus caecimuris as templates. Lanes 7-9 are out-of-genus templates, namely Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii respectively. Lane 10 is the negative control.

[0028] Figure 9 For the specific gene in Example 5 ECBG_02351 It is the single-PCR gel electrophoresis diagram of the primer pair of ECBG_02351 . In the figure, M is the DNA Marker. Lanes 1-6 are respectively using Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus casseliflavus, Enterococcus gallinarum, Enterococcus caecimuris as templates. Lanes 7-9 are out-of-genus templates, namely Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii respectively. Lane 10 is the negative control.

[0029] ​Figure 10 Single PCR gel electrophoresis diagram of the primer pair for the specific gene in Example 5 EGM181_RS15835 In the figure, M is the DNA Marker. Lanes 1-6 are respectively using Enterococcus casseliflavus, Enterococcus gallinarum, Enterococcus caecorum, Enterococcus casseliflavus, Enterococcus gallinarum, Enterococcus caecorum as templates. Lanes 7-9 are out-of-genus templates, namely Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii respectively. Lane 10 is the negative control.

[0030] Figure 11 Single PCR gel electrophoresis diagram of the primer pair for the specific gene in Example 5 DQL78_RS08525 In the figure, M is the DNA Marker. Lanes 1-6 are respectively using Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus casseliflavus, Enterococcus gallinarum, Enterococcus caecorum as templates. Lanes 7-9 are out-of-genus templates, namely Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii respectively. Lane 10 is the negative control.

[0031] Figure 12 Double PCR gel electrophoresis diagram of Example 6. In the figure, M is the DNA Marker. Lanes Q1-Q6 are 6 strains of Enterococcus casseliflavus. Lanes J1-P1 are respectively Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Serratia marcescens, Proteus mirabilis as negative controls. Lane N is the blank control.

[0032] Figure 13 Double PCR gel electrophoresis diagram of different concentrations of DNA templates in Example 7. In the figure, M is the DNA Marker. The DNA template concentrations of lanes 1-6 are 100 ng / μL, 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, 0.001 ng / μL respectively. The DNA template of lane 7 is Escherichia coli; A in the figure is Enterococcus casseliflavus, B is Enterococcus gallinarum, and C is Enterococcus caecorum.

[0033] Figure 14 Multiplex PCR gel electrophoresis diagram of Example 8. In the figure, M is the DNA Marker. Lane 1 is a mixed template of genomic DNA of three strains, Enterococcus faecium, Enterococcus faecalis and Enterococcus avium. Lanes 2-7 are respectively using Enterococcus avium, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus caecorum, Enterococcus casseliflavus as templates.

[0034] Figure 15This is the multiplex PCR gel electrophoresis diagram of template DNA with different concentrations in Example 9. In the figure, M is the DNA Marker, and the template DNA concentrations in lanes 1-7 are 100 ng / μL, 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, and 0.001 ng / μL respectively. Lane 7 uses Escherichia coli as the template as a negative control.

[0035] Figure 16 This is the multiplex PCR gel electrophoresis diagram of clinical samples in Example 10. In the figure, M is the DNA Marker, lane B is the mixed template of Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus caecorum, lanes Q1-Q6 are 6 strains of Enterococcus casseliflavus clinically, lane G is 1 strain of Enterococcus gallinarum clinically, lane C is 1 strain of Enterococcus caecorum clinically, and lane N is the blank control. Detailed implementation manners

[0036] The technical solutions of the present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the following embodiments are only the preferred embodiments of the present invention and should not be construed as limitations on the present invention. The protection scope of the present invention shall be subject to the content recorded in the claims. Modifications and substitutions made by those skilled in the art to the technical solutions of the present invention without creative efforts all fall within the protection scope of the present invention.

[0037] Example 1 (1) Obtain the single-copy core genes of Enterococcus from orthofinder; (2) Place the single-copy core genes of Enterococcus in LS-BSR, and through large-scale sequence alignment, analyze whether these genes exist in 35,104 Enterococcus genomes and do not exist in 1,426,688 non-Enterococcus genomes (common environmental microorganisms and human pathogens belonging to 49 genera); (3) Perform LS-BSR analysis on each conserved gene between Enterococcus and non-Enterococcus; (4) Compare the screened genes with the nucleotide database of NCBI to determine whether the gene belongs only to Enterococcus, and analyze the BSR values of the screened Enterococcus-specific genes.

[0038] The results are as Figure 1 shown. The rpmC gene is highly conserved in all Enterococcus genomes, with an average conservation rate of 88% and a minimum conservation rate of 83% ( Figure 1 A), rpmC and the BSR value of the Figure 1 gene in Enterococcus is 0.88 ( rpmCThe sequence of the gene is SEQ ID NO:1, and the protein ID is WP_002383858.1.

[0039] Example 2 (1) Analyze the genome of Enterococcus casseliflavus. Through LS-BSR, 666 highly conserved candidate identification genes were screened out (conservation rate: 90%); (2) Combine the alignment results with the average conservation rate (>95%) and minimum conservation rate (>90%) of the candidate identification genes in Enterococcus casseliflavus, as well as the average conservation rate (<10%) and maximum conservation rate (<40%) in other species and non-Enterococcus Figures 2 - 3 ); (3) Align the candidate identification genes with the NCBI database, and finally screen out the most conserved gene in the genomic DNA of Enterococcus casseliflavus as the specific molecular target: ECBG_02351 , with the sequence of SEQ ID NO:2 and protein ID of EEV40082.2.

[0040] Example 3 (1) Analyze the genome of Enterococcus gallinarum. Through LS-BSR, 165 highly conserved candidate identification genes were screened out (conservation rate: 90%); (2) Combine the alignment results with the average conservation rate (>95%) and minimum conservation rate (>90%) of the candidate identification genes in Enterococcus gallinarum, as well as the average conservation rate (<10%) and maximum conservation rate (<40%) in other species and non-Enterococcus Figures 4 - 5 ); (3) Align the candidate identification genes with the nt database in NCBI, and finally screen out the most conserved gene in the genomic DNA of Enterococcus gallinarum as the specific molecular target: EGM181_RS15835 , with the sequence of SEQ ID NO:3 and protein ID of WP_103300779.1.

[0041] Example 4 (1) Analyze the genome of Enterococcus caecorum. Through LS-BSR, 430 highly conserved candidate identification genes were screened out (conservation rate: 90%); (2) Combine the alignment results with the average conservation rate (>95%) and minimum conservation rate (>90%) of the candidate identification genes in Enterococcus caecorum, as well as the average conservation rate (<10%) and maximum conservation rate (<40%) in other species and non-Enterococcus Figures 6 - 7 ); (3)Align the candidate identified genes with the nt database in NCBI, and finally screen out the most conserved genes in the genomic DNA of Enterococcus cecorum as specific molecular targets: DQL78_RS08525 , with the sequence of SEQ ID NO:4 and the protein ID of WP_016251206.1.

[0042] Example 5 (1)Take the specific genes of Enterococcus identified in Example 1 rpmC , the specific genes of Enterococcus casseliflavus identified in Example 2 ECBG_02351 , the specific genes of Enterococcus gallinarum identified in Example 3 EGM181_ RS15835 , the specific genes of Enterococcus cecorum identified in Example 4 DQL78_RS08525 1, design primers for their sequences and send them to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The primer sequences are shown in Table 1; Table 1 PCR primer sequences

[0043] (2)Singleplex PCR: Respectively take rpmC the primer pair of the gene, ECBG_02351 the primer pair of the gene, EGM181_ RS15835 the primer pair of the gene and DQL78_RS08525 the primer pair of the gene, construct a PCR reaction system to verify their amplification specificity in different bacterial species. The PCR system is shown in Table 2, and the program is shown in Table 3; Table 2 PCR reaction system

[0044] Table 3 PCR reaction program

[0045] The results show that rpmC the primer pair of the gene can amplify specific fragments in all 6 Enterococcus strains, but there is no specific amplification template in other out-of-genus templates such as Escherichia coli and Staphylococcus aureus, indicating that rpmC the primer pair of the gene can specifically detect and identify Enterococcus strains, rpmC the gene is a specific molecular target for Enterococcus ( Figure 8 ). ECBG_02351 The primer pair of the gene can only amplify a single band in Enterococcus casseliflavus, and there is no specific amplification band in other Enterococcus strains and out-of-genus templates, indicating that ECBG_02351 the primer pair of the gene can specifically detect and identify Enterococcus casseliflavus ( Figure 9 ). EGM181_RS15835The primer pair of the gene can only amplify a unique band in Enterococcus gallinarum, and there are no specific amplification bands in other Enterococcus species and templates outside the genus, indicating that EGM181_RS15835 the primer pair of the gene can specifically detect and identify Enterococcus gallinarum ( Figure 10 ). DQL78_RS08525 The primer pair of the gene can only amplify a unique band in Enterococcus caecorum, and there are no specific amplification bands in other Enterococcus species and templates outside the genus, indicating that DQL78_RS08525 the primer pair of the gene can specifically detect and identify Enterococcus caecorum ( Figure 11 ).

[0046] Example 6 Mix the primer pair of the gene in the 4 pairs of primer pairs in Table 1 with the primer pair of any one of the three genes to prepare a primer composition, construct a duplex PCR reaction system, and perform duplex PCR amplification. The volume of the duplex PCR reaction is shown in Table 4, and the reaction program is the same as Table 3; the template in the duplex PCR reaction is the genomic DNA of Enterococcus flavum; and Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Serratia marcescens, Proteus mirabilis are used as negative controls rpmC Table 4 Duplex PCR reaction system

[0047] Figure 12 As can be seen from the duplex PCR detection can specifically detect and identify Enterococcus flavum. Since only one strain of Enterococcus gallinarum and Enterococcus caecorum was collected clinically, no separate verification was performed.

[0048] Example 7 Construct a multiplex PCR reaction system as in Example 6, and only dilute the concentration of the template DNA to 100 ng / μL, 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, 0.001 ng / μL respectively, and perform multiplex PCR reaction. The results are as Figure 13 shown that when the concentration of the template DNA ≥ 0.01 ng / μL, 2 specific bands can be accurately amplified.

[0049] Example 8 Mix the 4 pairs of primer pairs in Table 1 to prepare a primer composition, construct a multiplex PCR reaction system, and perform multiplex PCR amplification. The multiplex PCR reaction system is shown in Table 5, and the reaction program is the same as Table 3; Table 5 Multiplex PCR reaction system

[0050] The results show that Figure 14 after the primer pairs are combined to form a primer composition, Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus caecorum can be specifically detected and identified from the mixed templates of genomic DNA of multiple strains or the genomic DNA of a single strain.

[0051] Example 9 Genomic DNA of Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus caecorum was taken and mixed to obtain a template, which was respectively diluted to concentrations of 100 ng / μL, 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, and 0.001 ng / μL. Then, a multiplex PCR reaction system was configured according to Table 5 for PCR amplification. The results are as Figure 15 shown. When the concentration of genomic DNA ≥ 0.1 ng / μL, 4 specific bands can be accurately amplified.

[0052] Example 10 Urine, blood, or sputum samples clinically diagnosed as infected with Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus caecorum were collected, and their genomic DNA was extracted. A multiplex PCR reaction system was configured according to Table 5 for PCR amplification. The results are as Figure 16 shown. The designed 4 pairs of primers can accurately detect and identify Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus caecorum, indicating that rpmC gene, ECBG_02351 gene, EGM181_RS15835 gene, and DQL78_RS08525 gene can be applied to the detection and identification of Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus caecorum in clinical practice.

Claims

1. A specific molecular target for simultaneously detecting important pathogenic species within the genus Enterococcus, characterized in that: The specific molecular targets include an Enterococcus specific identification target rpmC and three enterococcal species-specific targets ECBG_ 02351 , EGM181_RS15835 , DQL78_RS08525 One or more of .

2. A specific molecular target for simultaneously detecting important pathogenic species within the genus Enterococcus according to claim 1, characterized in that: Said ECBG_02351 It is a specific target of Enterococcus pyogenes, and the nucleotide sequence is SEQ ID NO: 2 and its homologous sequence.

3. The specific molecular target for simultaneously detecting important pathogenic species within the genus Enterococcus according to claim 1, characterized in that: Said EGM181_RS15835 It is a specific target of Enterococcus gallinarum, and the nucleotide sequence is SEQ ID NO: 3 and its homologous sequence.

4. The method according to claim 1, wherein: Said DQL78_RS08525 It is a specific target for cecal Enterococcus, and the nucleotide sequence is SEQ ID NO: 4 and its homologous sequence.

5. The specific molecular target for simultaneously detecting important pathogenic species within the genus Enterococcus according to claim 1, characterized in that: Said rpmC The nucleotide sequence is SEQ ID NO: 1 and its homologous sequences.

6. A specific molecular target for simultaneously detecting important pathogenic species within the genus Enterococcus according to any one of claims 2 to 5, characterized in that: The homologous sequence refers to a sequence identity of more than 85%.

7. A kit for simultaneously detecting important pathogenic species within the genus Enterococcus, characterized in that: The kit comprises a specific primer pair composition, a PCR reaction solution, a positive control and a negative control.

8. A kit for simultaneously detecting important pathogenic species within the genus Enterococcus according to claim 7, characterized in that: The specific primer pair composition includes an Enterococcus-specific identification target rpmC Primer pair, specific target for Enterococcus pyogenes ECBG_02351 Primer pair, specific target for Enterococcus gallinarum EGM181_RS15835 Primer pair, cecal Enterococcus specific target DQL78_RS08525 Two or more pairs of primer pairs.

9. A kit for simultaneously detecting important pathogenic species within the genus Enterococcus according to claim 8, characterized in that: Said rpmC The sequences of the primer pairs are SEQ ID NO: 5 and SEQ ID NO: 6 respectively; ECBG_02351 The sequences of the primer pair are SEQ ID NO: 7 and SEQ ID NO: 8; EGM181_RS15835 The sequences of the primer pair are SEQ ID NO: 9 and SEQ ID NO: 10; DQL78_RS08525 The sequences of the primer pair are SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

10. A detection method of a kit for simultaneously detecting important pathogenic species of the genus Enterococcus according to any one of claims 7 to 9, characterized in that: The steps include: (1) Extract DNA from the sample to be tested; (2) using the extracted DNA as a template, establishing a double PCR reaction system with a specific primer pair combination and a PCR reaction solution, and conducting a PCR reaction; (3) Determine the reaction results: The specific fragments with sizes of 161 bp, 285 bp, 629 bp and 417 bp were amplified, indicating that the sample to be tested contained Enterococcus pyogenes, Enterococcus gallinarum and Enterococcus caecum. The specific fragments obtained by amplification are 161 bp, 285 bp, and 629 bp, respectively, indicating that the sample to be tested contains Enterococcus foetida and Enterococcus gallinarum. If the specific fragments with sizes of 161 bp, 285 bp and 417 bp are obtained by amplification, the sample to be tested contains Enterococcus leucoderma and Enterococcus caecum; The specific fragments obtained by amplification are 161 bp, 629 bp and 417 bp respectively, indicating that the sample to be tested contains Enterococcus gallinarum and Enterococcus caecum. If the amplified specific fragments are 161 bp and 285 bp in size, the sample to be tested contains Enterococcus fusiformis; If the amplified specific fragments are 161 bp and 629 bp respectively, the sample to be tested contains Enterococcus gallinarum; If the amplified specific fragments are 161 bp and 417 bp in size, the sample to be tested contains cecal cocci; If a specific fragment with a size of 161 bp is amplified, the sample to be tested contains Enterococcus, but does not contain Enterococcus pyogenes, Enterococcus gallinarum and Enterococcus caecum; If no specific fragment is amplified, the sample to be tested does not contain any pathogenic bacteria of the genus Enterococcus.

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