Target gene, primer pair, kit and detection method for detecting multi-animal streptococcus and application

Abstract

The invention discloses a target gene, a primer pair, a kit and a detection method for detecting multi-animal streptococcus and application, and relates to the technical field of bioengineering. The nucleotide sequence of the target gene for detecting the multi-animal streptococcus is as shown in SEQ ID NO: 1; the sequences of the primer pair designed based on the target gene are shown as SEQ ID NO: 2 and SEQ ID NO: 3; the fluorescent quantitative PCR kit for detecting the multi-animal streptococcus comprises a primer pair and a fluorescent quantitative PCR reaction premixed solution, the fluorescent quantitative PCR method for detecting the multi-animal streptococcus comprises the following steps: establishing a standard curve between a multi-animal streptococcus amplification CT value and a template copy number, and quantifying the multi-animal streptococcus in a sample to be detected through the standard curve. Clinical application proves that multi-animal streptococcus in mouse lung tissues and clinical swab samples can be effectively detected. The invention provides reliable technical support for rapid diagnosis and epidemiological investigation of multi-animal streptococcus.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method and application for detecting target genes, primer pairs, kits, and detection methods of various animal streptococci. Background Technology

[0002] Multiple animal streptococci ( Streptococcus pluranimalium It is a zoonotic pathogen that can cause respiratory infections, septicemia, and arthritis in animals such as horses, pigs, and dogs, posing a threat to the development of animal husbandry and public health security.

[0003] However, there are significant shortcomings in efficient detection technologies for this pathogen, hindering rapid response and effective control of outbreaks. Currently, the identification of Streptococcus multifiliis mainly relies on traditional bacterial isolation and culture methods, often followed by 16S rRNA gene sequencing for confirmation. This method is time-consuming, taking 24 to 48 hours, and has low sensitivity, making it difficult to meet the detection needs of low bacterial loads in clinical samples. Quantitative real-time PCR (qPCR) technology combines the high sensitivity of PCR, the high specificity of fluorescent labeling, and real-time quantification capabilities, and has been successfully applied to the rapid detection of various pathogens. For example, this technology has been used for species-level identification and serotyping of Streptococcus suis, demonstrating excellent performance. However, despite the maturity of this technology, specific qPCR detection methods for Streptococcus multifiliis remain lacking. This technological gap directly leads to low efficiency in outbreak monitoring and epidemiological investigations. Therefore, there is an urgent need to develop a rapid, highly sensitive, and specific quantitative detection scheme for Streptococcus multifiliis. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art or related art.

[0005] Therefore, in a first aspect, the present invention provides a target gene for detecting multiple animal streptococci, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0006] In a second aspect, the present invention provides a primer pair designed based on the target gene, wherein the specific sequence of the upstream primer is shown in SEQ ID NO:2 and the specific sequence of the downstream primer is shown in SEQ ID NO:3.

[0007] A third aspect of the present invention provides a real-time quantitative PCR kit for detecting multiple animal streptococci, comprising: the primer pair and a real-time quantitative PCR reaction premix.

[0008] Furthermore, the kit also includes a standard plasmid; wherein the standard plasmid contains the target gene.

[0009] Furthermore, the nucleotide sequence of the standard plasmid is shown in SEQ ID NO:4.

[0010] Furthermore, the method for constructing the standard plasmid includes: The primer pair was subjected to PCR amplification to obtain the amplification product; after purification, the amplification product was seamlessly cloned and ligated with plasmid pMD-19T, and the resulting ligation product was transformed into Escherichia coli DH5α to extract the standard plasmid pMD19-T-rbsR.

[0011] Furthermore, the real-time PCR reaction system of the kit is as follows: 10 μL SYBR Green Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 2 μL template, and 7 μL RNase-free water; wherein the template is the DNA of the sample to be tested or the standard plasmid. The real-time PCR reaction conditions for the kit are: 95℃ for 2 min; 95℃ for 15 s, 60℃ for 1 min, for 40 cycles.

[0012] A fourth aspect of the present invention provides a method for detecting multiple animal streptococci using real-time quantitative PCR, the method comprising: Establishment of standard curve: Using the standard plasmid as a DNA template, the primer pair was used for real-time PCR amplification, and a standard curve was plotted between the amplification CT value of Streptococcus multifiliis and the template copy number. Sample detection: Using the DNA of the sample to be tested as a template, the primer pair is used to perform real-time PCR amplification to obtain the CT value of the sample to be tested, and then the quantitative result of multiple animal streptococci in the sample to be tested is calculated according to the standard curve. The method is used for the diagnosis and treatment of non-disease conditions.

[0013] Furthermore, the standard curve is Y = -3.402lg(x) + 40.50, and the correlation coefficient R0 is... 2 =0.999.

[0014] A fifth aspect of the invention provides the application of the target gene, the primer pair, the kit, or the method thereof in identifying multi-animal streptococci and differentiating streptococcal species.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: This invention successfully screened the specific target gene rbsR as a detection target by comparing the whole genome sequences of Streptococcus multifiliis and its closely related streptococci. Specific primers were designed for this gene, the reaction system was optimized, and a standard plasmid was constructed to plot a standard curve, thus establishing a rapid, sensitive, and specific real-time quantitative PCR (qPCR) detection method and corresponding kit for Streptococcus multifiliis. Results showed that this method can detect Streptococcus multifiliis within a 3×10⁻⁶ range. 1 Copy / μL to 3×10 7 Good linearity in the copy / μL range (R 2 The detection sensitivity is as high as 30 copies / μL (=0.999), and there is no cross-reactivity with common bacteria such as Escherichia coli, Streptococcus suis, and Streptococcus agalactiae. The within-group and between-group coefficients of variation are both less than 1.5%, indicating good repeatability. Clinical applications have confirmed that this invention can effectively detect multiple animal streptococci in mouse lung tissue and clinical swab samples. This invention provides reliable technical support for the rapid diagnosis and epidemiological investigation of multiple animal streptococci. Attached Figure Description

[0016] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of this application. In the drawings: Figure 1 This is a melting curve after fluorescence quantitative PCR amplification provided in an embodiment of the present invention; Figure 2 This is a standard curve between amplification CT values ​​of multiple animal streptococci provided in the embodiments of the present invention; Figure 3 The image shows a specific experimental amplification diagram provided in the embodiments of the present invention; wherein, sample 1 is a multi-animal streptococcus, samples 2-6 are Escherichia coli, Streptococcus suis, Streptococcus agalactiae, Streptococcus lactis, and Enterococcus faecalis strains, respectively, and sample 7 is a negative control; Figure 4 This is a detection diagram of Streptococcus multifiliis in mouse lung tissue provided in an embodiment of the present invention; wherein, sample 1 is a positive control, samples 2-4 are mouse lung tissue, and sample 5 is a negative control; Figure 5 This is a clinical bovine nasal swab image for detecting multiple animal streptococci provided in an embodiment of the present invention; wherein, samples 1-10 are calf swabs, sample 11 is a positive control, and sample 12 is a negative control. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0018] In the first aspect of the embodiments of the present invention, a target gene for detecting Streptococcus equi subsp. zooepidemicus is provided, and the nucleotide sequence of the target gene is as shown in SEQ ID NO: 1.

[0019] In the embodiments of the present invention, the complete genomic sequences of 13 strains of Streptococcus equi subsp. zooepidemicus in the National Center for Biotechnology Information (NCBI) gene sequence database were compared with the complete genomic sequences of 123 other Streptococcus bacteria in groups such as mutans, pyogenic, suis, mitis, anginosus, and pluranimalium. It was found that there is a conserved gene in Streptococcus equi subsp. zooepidemicus rbsR , and this gene does not exist in other related Streptococcus bacteria. The nucleotide sequence of this gene was uploaded to NCBI for BLASTN comparison, and the matching rate between the Streptococcus equi subsp. zooepidemicus genome and this sequence segment was more than 96.95%, and the coverage rate was 100%. However, there was no matching degree in other related Streptococcus genomes, which proved that this gene has good sequence specificity. Therefore, the rbsR gene (as shown in SEQ ID NO: 1) was used as the target gene for detecting Streptococcus equi subsp. zooepidemicus.

[0020] In the second aspect of the embodiments of the present invention, a primer pair capable of specifically amplifying the rbsR gene in Streptococcus equi subsp. zooepidemicus was designed, and a primer pair designed based on the target gene of Streptococcus equi subsp. zooepidemicus was provided. The specific sequence of the upstream primer of the primer pair is as shown in SEQ ID NO: 2, and the specific sequence of the downstream primer is as shown in SEQ ID NO: 3. [[ID= (12]]

[0021] In the third aspect of the embodiments of the present invention, a fluorescence quantitative PCR kit for detecting Streptococcus equi subsp. zooepidemicus is provided, including: the above-mentioned primer pair and a fluorescence quantitative PCR reaction premix; Among them, the primer pair is used for fluorescence quantitative PCR amplification of the DNA of the待测样品 (to be determined sample) or the standard plasmid.

[0022] In some embodiments, the fluorescence quantitative PCR kit for detecting Streptococcus equi subsp. zooepidemicus further includes: a standard plasmid; among them, the standard plasmid is used to construct a standard curve for quantitatively detecting Streptococcus equi subsp. zooepidemicus, and the standard plasmid contains the target gene of Streptococcus equi subsp. zooepidemicus.

[0023] In some embodiments, the nucleotide sequence of the standard plasmid is as shown in SEQ ID NO: 4.

[0024] In some embodiments, the standard plasmid was constructed by integrating the amplification product of the above-mentioned primer pair into the plasmid pMD-19T.

[0025] It should be noted that the term "待测样品" in the translation is a placeholder for the specific Chinese term that needs to be determined according to the context. If there is a more specific and accurate English expression in the original context, it should be used for replacement.Optionally, methods for constructing standard plasmids include: The primer pairs described above were used for PCR amplification to obtain the amplification product. After purification, the amplification product was seamlessly cloned and ligated with plasmid pMD-19T. The ligation product was transformed into Escherichia coli DH5α and the standard plasmid pMD19-T-rbsR was extracted.

[0026] In some embodiments, the real-time PCR reaction system of the multi-animal streptococcus real-time PCR kit is as follows: 10 μL SYBR Green Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 2 μL of DNA template or standard plasmid template of the sample to be tested, and 7 μL of RNase-free water.

[0027] In some embodiments, the fluorescence quantitative PCR reaction conditions for the multi-animal streptococcus fluorescence quantitative PCR kit are: 95℃ for 2 min; 95℃ for 15 s, 60℃ for 1 min, for 40 cycles.

[0028] A fourth aspect of the present invention provides a method for detecting multiple animal streptococci using real-time quantitative PCR, comprising: Establishment of standard curve: Using the above standard plasmid as DNA template, the above primer pair was used for real-time PCR amplification, and a standard curve was plotted between the amplified CT value of Streptococcus multifiliis and the template copy number. Sample detection: Using the DNA of the sample to be tested as a template, the above primer pair was used to perform real-time PCR amplification to obtain the CT value of the sample to be tested, and then the quantitative result of multiple animal streptococci in the sample to be tested was calculated according to the standard curve. The above methods are used for the diagnosis and treatment of non-disease-related conditions.

[0029] In some embodiments, the standard curve is Y = -3.402lg(x) + 40.50, and the correlation coefficient R0 is... 2 =0.999.

[0030] The quantitative real-time PCR method for detecting multiple animal streptococci provided in this embodiment of the invention, at 3×10 1 Copy / μL to 3×10 7 copy / μL (R) 2 The method exhibits good linearity within the range of 0.999, with a detection sensitivity as high as 30 copies / μL, and shows no cross-reactivity with common bacteria such as Escherichia coli, Streptococcus suis, and Streptococcus agalactiae. The coefficients of variation within and between groups are both less than 1.5%. This demonstrates that the method has a wide linear range, high sensitivity, strong specificity, and good repeatability.

[0031] A fifth aspect of the present invention provides the application of the above-mentioned target gene, primer pair, kit, or quantitative real-time PCR method for detecting multiple animal streptococci in identifying multiple animal streptococci and differentiating streptococcal species.

[0032] The quantitative real-time PCR method for multiple animal streptococci established in this invention is based on the SYBR Green I dye method. The specificity of the amplified products is further confirmed by melting curve analysis. It can effectively distinguish closely related streptococci such as Streptococcus agalactiae, Streptococcus lactis, and common bacteria such as Escherichia coli and Streptococcus suis. The amplification efficiency is high. Therefore, this method can be used to identify multiple animal streptococci and distinguish streptococci, and has good specificity, sensitivity and repeatability.

[0033] Clinical applications have confirmed that the quantitative real-time PCR method for multiple animal streptococci established in this invention can effectively detect multiple animal streptococci in mouse lung tissue and clinical swab samples. The quantitative real-time PCR method for multiple animal streptococci established in this invention provides reliable technical support for the rapid diagnosis and epidemiological investigation of multiple animal streptococci.

[0034] Unless otherwise specified, the materials, reagents, instruments, and methods used in the following examples are all conventional materials, reagents, instruments, and methods in the art and are commercially available.

[0035] Example 1: Quantitative Real-Time PCR Detection Method for Multiple Animal Streptococci 1. Materials and Methods 1.1 Materials 1.1.1 Bacteria Multiple animal streptococci ( Streptococcus pluranimalium Escherichia coli, Streptococcus suis, Streptococcus agalactiae, Streptococcus lactis, and Enterococcus faecalis were all preserved in this laboratory.

[0036] 1.1.2 Main Reagents and Instruments Nucleic acid extraction kit: QIAamp DNA Mini Kit; SYBR Green Master Mix: ChamQ Universal SYBR qPCR Master Mix; Real-time PCR instrument: LightCycler® 96.

[0037] 1.2 Methods 1.2.1 Primer pairs and their amplification products Primers were designed based on the conserved region of the rbsR gene, using the genome sequences of *Streptococcus multifiliis* published in GenBank. Primer pair sequences are shown in Table 1.

[0038] Table 1 Primer pairs and amplification products for quantitative real-time PCR of Streptococcus multifiliis.

[0039] 1.2.2 Preparation of standard plasmid pMD19-T-rbsR The steps for constructing the recombinant positive standard plasmid pMD19-T-rbsR by integrating the product obtained from PCR amplification using primer pairs in step 1.2.1 into plasmid pMD19-T are as follows: The PCR amplification product is transferred to a 1.5 mL centrifuge tube, followed by agarose gel electrophoresis, gel cutting, and gel recovery to obtain the purified PCR product; using a commercially available seamless cloning kit, the purified PCR amplification product and plasmid pMD19-T are integrated into the plasmid pMD19-T-rbsR. Seamless cloning was performed using 19T; the obtained seamless clone product was transformed into Escherichia coli DH5α using a chemical transformation method; the recombinant positive standard plasmid pMD19-T-rbsR was extracted from Escherichia coli DH5α using a commercial plasmid extraction kit and used in subsequent experiments.

[0040] 1.2.3 Real-time PCR reaction system and reaction conditions The PCR reaction system consisted of: 10 μL of SYBR Green Master Mix, 0.5 μL each of forward and reverse primers (10 μM), 2 μL of template, and RNase-free water to a final volume of 20 μL.

[0041] (When testing the sample, the DNA of the sample is used as a template; in the positive control, the standard plasmid pMD19-T-rbsR is used as a template.) The reaction conditions were: 95℃ for 2 min; 95℃ for 15 s, 60℃ for 1 min, for 40 cycles.

[0042] 1.2.4 Establishment of the Standard Curve The standard plasmid pMD19-T-rbsR was used as a DNA template and serially diluted 10-fold with RNase-free ddH2O to prepare standard templates of different concentrations. Fluorescent PCR amplification was performed using the upstream primer (Primer F) SEQ ID NO:2 and the downstream primer (Primer R) SEQ ID NO:3, following the reaction conditions and reaction system recommended in step 1.2.3. SYBR GreenI fluorescent dye qPCR amplification was performed, and a standard curve was plotted using GraphPad Prism 9.0.

[0043] 1.2.5 Specificity test The specificity of this method was verified by detecting various animal streptococci, as well as strains of Escherichia coli, Streptococcus suis, Streptococcus agalactiae, Streptococcus lactis, and Enterococcus faecalis.

[0044] 1.2.6 Repeatability Verification of the Method Using the optimized reaction conditions, amplification was performed with standard plasmids of different concentrations as templates. Three replicates were set for each concentration. The mean Ct value, standard deviation, and coefficient of variation within and between groups were calculated.

[0045] 1.2.7 Method Application The method was used to detect lung tissue and clinical swab samples from mice infected with multiple animal streptococci, and the practicality of the method was evaluated.

[0046] 2 Results 2.1 Establishment of the standard curve like Figure 1 As shown, the melting curve exhibits a single, specific peak, confirming the amplification specificity of this method. Figure 2 As shown, the recombinant positive standard plasmid pMD19-T-rbsR was diluted 10-fold to a concentration of 3 × 10⁻⁶. 1 Copy / μL to 3×10 7 For each concentration of standard, three replicates were performed for quantitative fluorescence reaction to obtain a standard curve. The standard equation is: Y = 3.402lg(x) + 40.50, correlation coefficient R 2 =0.999, indicating that the recombinant positive standard plasmid pMD19-T-rbsR has a good linear relationship within this concentration range. Furthermore, the detection method provided in this embodiment of the invention has a detection limit of 30 copies / μL for Streptococcus multifiliis DNA, demonstrating high sensitivity.

[0047] 2.2 Specificity Experiment Results The real-time quantitative PCR detection method is specifically used for detection, and the results are as follows: Figure 3 As shown, the amplification curves of Escherichia coli, Streptococcus suis, Streptococcus agalactiae, Streptococcus lactis, and Enterococcus faecalis were all linear with no amplification, while the amplification curve of Streptococcus multifiliis was obvious (Ct value of about 24) when Streptococcus multifiliis was used as a template, indicating that the established real-time PCR detection method has good specificity.

[0048] 2.3 Repeatability Validation of the Method The plasmid standards at the following concentrations were detected using the established quantitative real-time PCR method, as shown in Table 2.

[0049] Table 2. Analysis of intra-batch and inter-batch coefficients of variation (CV) of standards at different concentrations.

[0050] As can be seen from Table 2, the coefficients of variation within and between plasmid standard groups are both less than 1.5%, proving that the method has good reproducibility.

[0051] 2.4 Method Application like Figure 4 As shown, the established quantitative real-time PCR method for detecting this bacterium (1×10⁻⁶) was used. 8 Three lung tissue samples were collected from mice infected with CFU, and all samples tested positive. Meanwhile, as... Figure 5 As shown, this method was used to test 10 nasal swabs from clinical calves, and sample 5 was positive for multiple animal streptococci. These two results indicate that this method can be effectively used for the detection of infection models in experimental animals and for the diagnosis of clinical samples.

[0052] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting target genes of multiple animal streptococci, characterized in that, The nucleotide sequence of the target gene is shown in SEQ ID NO:

1.

2. A primer pair designed based on the target gene described in claim 1, characterized in that, The specific sequence of the upstream primer in the primer pair is shown in SEQ ID NO:2, and the specific sequence of the downstream primer is shown in SEQ ID NO:

3.

3. A real-time quantitative PCR kit for detecting multiple animal streptococci, characterized in that, include: The primer pair and premixed solution for real-time PCR reaction as described in claim 2.

4. The reagent kit according to claim 3, characterized in that, The kit also includes a standard plasmid; wherein the standard plasmid contains the target gene as described in claim 1.

5. The reagent kit according to claim 4, characterized in that, The nucleotide sequence of the standard plasmid is shown in SEQ ID NO:

4.

6. The reagent kit according to claim 4, characterized in that, The method for constructing the standard plasmid includes: The primer pair described in claim 2 was subjected to PCR amplification to obtain the amplification product; after purification, the amplification product was seamlessly cloned and ligated with plasmid pMD-19T, and the resulting ligation product was transformed into Escherichia coli DH5α to extract the standard plasmid pMD19-T-rbsR.

7. The reagent kit according to claim 3, characterized in that, The real-time PCR reaction system of the kit is as follows: 10 μL SYBR Green Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 2 μL template, and 7 μL RNase-free water; wherein the template is the DNA of the sample to be tested or the standard plasmid. The real-time PCR reaction conditions for the kit are: 95℃ for 2 min; 95℃ for 15 s, 60℃ for 1 min, for 40 cycles.

8. A real-time quantitative PCR method for detecting multiple animal streptococci, characterized in that, The method includes: Establishment of standard curve: Using the standard plasmid described in claim 3 as a DNA template, fluorescent quantitative PCR amplification was performed using the primer pair described in claim 2, and a standard curve between the amplification CT value of multiple animal streptococci and the template copy number was plotted. Sample detection: Using the DNA of the sample to be tested as a template, the primer pair described in claim 2 is used to perform real-time PCR amplification to obtain the CT value of the sample to be tested, and then the quantitative result of multiple animal streptococci in the sample to be tested is calculated according to the standard curve. The method is used for the diagnosis and treatment of non-disease conditions.

9. The method for detecting multiple animal streptococci using real-time PCR according to claim 8, characterized in that, The standard curve is Y = -3.402lg(x) + 40.50, and the correlation coefficient R0 is... 2 =0.

999.

10. The use of the target gene of claim 1, the primer pair of claim 2, the kit of any one of claims 3-7, or the method of any one of claims 8-9 in the identification of multi-animal streptococci and the differentiation of streptococcal genus.

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