A dual fluorescence quantitative PCR kit for simultaneous detection of Streptococcus pasteurianus and Streptococcus suis

Abstract

The present invention provides a dual fluorescent quantitative PCR kit for simultaneously detecting Streptococcus pasteurianus and Streptococcus suis, belonging to the field of bacterial detection. The kit includes a primer pair and a probe. The primer pair and probe for Streptococcus pasteurianus include SP-3230F, SP-3230R and SP-probe, and the nucleotide sequence is shown in SEQ ID NO: 1-3; the primer pair and probe for Streptococcus suis include SS-recNF, SS-recNR and SS-probe, and the nucleotide sequence is shown in SEQ ID NO: 4-6. The kit of the present invention has the advantages of high sensitivity, high specificity, low pollution and real-time detection, and provides reliable technology and products for the early diagnosis, monitoring, early warning and prevention of clinical streptococcal disease in pigs in farms.

Description

Technical Field

[0001] The invention belongs to the field of bacterial detection, and in particular relates to a dual fluorescence quantitative PCR kit for simultaneously detecting Streptococcus pasteurianus and Streptococcus suis. Background Art

[0002] Streptococcus pasteurianus ( Streptococcus pasteurianus ) is a new zoonosis. It can cause meningitis and septicemia in nine species of animals, including geese, turkeys, and pigs. It can also cause illness or death in humans, causing endocarditis, urinary tract infections in newborns, the elderly, and immunocompromised individuals, and intrauterine infections in pregnant women. It may also be associated with gastrointestinal malignancies in humans. Researchers have confirmed for the first time that Streptococcus pasteurianus can cause meningitis in pigs, identifying it as a new pathogen of streptococcal disease in swine. Preliminary experiments have demonstrated that S. pasteurianus can be found in the tonsils and hilar lymph nodes of healthy pigs, and that both S. suis and S. pasteurianus can be detected in the same tonsil.

[0003] Streptococcus suis ( Streptococcus suis S. spp. (SS) is a major pathogen in pigs, primarily colonizing the upper respiratory tract, particularly the pharynx and tonsils. It can cause meningitis, arthritis, sepsis, and sudden death, resulting in severe economic losses for the pig industry. It is also a zoonosis, causing meningitis, sepsis, and even death in humans.

[0004] Both Streptococci are the pathogens of Streptococcus suis, and clinical co-infections occur. This has a multifaceted impact on the aquaculture industry, including not only direct economic losses but also challenges to drug resistance, public health, and sustainable development. Currently, the species-specific genes of S. pasteurianus have not been identified, and sensitive and specific molecular detection methods for this bacterium are scarce, resulting in poor detection results. Therefore, providing a dual fluorescence quantitative PCR kit capable of simultaneously detecting S. pasteurianus and S. suis is an urgent need for researchers in this field. Summary of the Invention

[0005] The purpose of the present invention is to provide a dual fluorescence quantitative PCR kit for the simultaneous detection of Streptococcus pasteurianus and Streptococcus suis. The kit has the advantages of high sensitivity, high specificity and real-time detection, and provides reliable technology and products for the early diagnosis, monitoring, early warning and prevention of clinical Streptococcus suis disease in farms and animal food safety inspection.

[0006] The purpose of the present invention is achieved by adopting the following technical solutions:

[0007] A dual fluorescence quantitative PCR kit for simultaneously detecting Streptococcus pasteurianus and Streptococcus suis, comprising a primer pair for Streptococcus pasteurianus, a probe SP-probe for Streptococcus pasteurianus, a primer pair for Streptococcus suis, and a probe SS-probe for Streptococcus suis; the primer pair for Streptococcus pasteurianus comprises a forward primer SP-3230F and a reverse primer SP-3230R, the sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the probe SP-probe for Streptococcus pasteurianus has a fluorescent group VIC at its 5' end and a quenching group BHQ1 at its 3' end, and its nucleotide sequence is shown in SEQ ID NO: 3; the primer pair for Streptococcus suis comprises a forward primer SS-recNF and a reverse primer SS-recNR, the sequences of which are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively. NO:5 shows; the probe SS-probe for Streptococcus suis has a fluorescent group FAM at the 5' end and a quenching group BHQ1 at the 3' end, and its nucleotide sequence is shown in SEQ ID NO:6.

[0008] In a preferred technical solution, the concentration of each primer and probe in the kit is 8-12 nmol / L.

[0009] In the present invention, the kit further comprises a plasmid standard product of Streptococcus pasteurianus and a plasmid standard product of Streptococcus suis, wherein the plasmid standard product of Streptococcus pasteurianus and the plasmid standard product of Streptococcus suis are respectively E8M05_RS03230 Gene fragments and Streptococcus suis recN The gene fragment is inserted into the vector.

[0010] In the present invention, the concentrations of the Streptococcus pasteurianus plasmid standard and the Streptococcus suis plasmid standard are both 1×10 9 copies / μL-1×10 2 copies / μL.

[0011] The present invention also provides a method for detecting Streptococcus pasteurianus and Streptococcus suis using the kit for non-diagnostic purposes, comprising the following steps:

[0012] (1) Using the total genomic DNA extracted from the sample to be tested as a template, a dual Taqman fluorescence quantitative PCR reaction was performed using primer pairs and probes targeting Streptococcus pasteurianus and Streptococcus suis. The fluorescence channels of the qPCR instrument were set as follows: channel 1: FAM, channel 2: VIC;

[0013] (2) When the Ct value in channel 1 is ≤35 and there is a specific amplification curve, it is judged as positive, that is, the sample contains Streptococcus suis; when the Ct value in channel 2 is ≤35 and there is a specific amplification curve, it is judged as positive, that is, the sample contains Streptococcus pasteurianus.

[0014] In the present invention, the dual Taqman fluorescence quantitative PCR reaction system is as follows: fluorescence quantitative enzyme reaction solution LTM012 17.45 μL, forward primer SP-3230F 0.5 μL, reverse primer SP-3230R 0.5 μL, probe SP-probe 0.25 μL, forward primer SS-recNF 0.4 μL, reverse primer SS-recNR 0.4 μL, probe SS-probe 0.5 μL, template 5 μL.

[0015] In the present invention, the dual Taqman fluorescence quantitative PCR reaction amplification procedure is as follows: incubation at 37°C for 2 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, for a total of 40 cycles.

[0016] The beneficial effects of the present invention are as follows:

[0017] (1) The present invention can simultaneously detect Streptococcus pasteurianus and Streptococcus suis in one reaction tube, providing a simple, fast, efficient and low-cost method for the detection of the pathogen of suis streptococcal disease.

[0018] (2) The dual TaqMan probe qPCR detection method established in the present invention has a high sensitivity for detecting each target pathogen. It has been verified that the sensitivity of the kit of the present invention for Streptococcus suis can reach 10 1 copies / μL, Streptococcus pasteurianus 1×10 2 copies / μL, with good specificity and repeatability.

[0019] (3) The kit provided by the present invention provides a reliable basis for the prevention and control of this type of disease, thereby greatly reducing the workload of single-plex qPCR detection and significantly improving work efficiency.

[0020] (4) The kit of the present invention has good specificity and repeatability, and has high sensitivity. It can be directly used for clinical testing and has the advantages of rapid, efficient and real-time detection. It provides reliable technology and products for the early diagnosis, monitoring, early warning and prevention of clinical swine streptococcal disease in farms and animal food safety inspection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the BLAST alignment result of the target sequence of the universal primer-probe combination of Streptococcus suis.

[0022] Figure 2These are the specificity test results for the three pairs of Streptococcus pasteurianus primer-probe compositions in Example 1, each paired with a Streptococcus suis primer-probe composition. A shows the combined detection result of Streptococcus pasteurianus primer-probe composition 1 and a Streptococcus suis primer-probe composition; B shows the combined detection result of Streptococcus pasteurianus primer-probe composition 2 and a Streptococcus suis primer-probe composition; and C shows the combined detection result of Streptococcus pasteurianus primer-probe composition 3 and a Streptococcus suis primer-probe composition. Figure 2 The right ends of the curves in A, B, and C correspond to the strain names on the right from top to bottom.

[0023] Figure 3 The results of the reliability experiment of the kit of the present invention are shown, wherein: A is the fluorescence amplification curve of the Streptococcus pasteurianus plasmid standard at various concentrations, the abscissa is the CT value, and the ordinate is the fluorescence intensity; B is the fluorescence amplification curve of the Streptococcus suis plasmid standard at various concentrations, the abscissa is the CT value, and the ordinate is the fluorescence intensity; C is the standard curve of Streptococcus pasteurianus, the abscissa is 1gC, C is the concentration of the Streptococcus pasteurianus plasmid standard, the unit of C is copies / μL, and the ordinate is the CT value; D is the standard curve of the Streptococcus suis plasmid standard at various concentrations, the abscissa is 1gC, C is the concentration of the Streptococcus suis plasmid standard, the unit of C is copies / μL, and the ordinate is the CT value.

[0024] Figure 4 The sensitivity test results of the kit of the present invention for detecting a single pathogen in Example 2 are shown in Figure 2. The concentrations of the plasmid standards corresponding to the curves from left to right (8 to 1) are 10 8 -10 1 copies / μL, the horizontal axis is the CT value, and the vertical axis is the fluorescence intensity.

[0025] Figure 5 The specificity test results of the kit of the present invention are shown in FIG. 1 , where the horizontal axis represents the CT value and the vertical axis represents the fluorescence intensity.

[0026] Figure 6 Results of the test kit of the present invention for detecting various serotypes of Streptococcus suis. The green specific amplification curve corresponds to each serotype of Streptococcus suis. The blue specific amplification curve corresponds to the positive control. DETAILED DESCRIPTION

[0027] The following is a detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned contents of the present invention fall within the scope of the present invention. Unless otherwise specified, the following examples are all completed using conventional existing technologies.

[0028] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art.

[0029] Example 1 Dual Fluorescence Quantitative PCR Kit for Simultaneous Detection of Streptococcus pasteurianus and Streptococcus suis and Detection Method Thereof

[0030] 1. Dual fluorescence quantitative PCR kit for simultaneous detection of Streptococcus pasteurianus and Streptococcus suis

[0031] A dual fluorescence quantitative PCR kit for simultaneously detecting Streptococcus pasteurianus and Streptococcus suis (abbreviated as the kit of the present invention) comprises primers, probes and plasmid standards.

[0032] (1) Primers and probes

[0033] By analyzing and comparing the gene sequences of Streptococcus pasteurianus and Streptococcus suis, primers and probes were designed targeting multiple target genes from each of these two strains. Using nucleic acids from these strains as templates, dual Taqman fluorescence quantitative PCR was used to identify primers and probes that could simultaneously and accurately identify both strains with high sensitivity. First, using Beacon Designer 7 software, dozens of primer and probe pairs were designed targeting the gene sequences of Streptococcus pasteurianus and Streptococcus suis. The feasibility of different primer combinations was analyzed in order to identify the optimal primer and probe combination for simultaneous detection of both strains. Because individual primer pairs can exhibit complementarity and primer-dimer formation, as well as interactions between primers, between primers and probes, and between probes, such as hairpin formation and dimerization, finding highly sensitive probes and primers for simultaneous detection of both strains is challenging. Through a lot of creative work, we finally obtained the forward primer SP-3230F, reverse primer SP-3230R, and probe SP-probe for Streptococcus pasteurianus, and the forward primer SS-recNF, reverse primer SS-recNR, and probe SS-probe for Streptococcus suis.

[0034] The kit of the present invention includes a primer pair for Streptococcus pasteurianus, a probe SP-probe for Streptococcus pasteurianus, a primer pair for Streptococcus suis, and a probe SS-probe for Streptococcus suis. The primer pair for Streptococcus pasteurianus includes a forward primer SP-3230F and a reverse primer SP-3230R, the sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The probe SP-probe for Streptococcus pasteurianus has a fluorescent group VIC at its 5' end and a quencher group BHQ1 at its 3' end, and its nucleotide sequence is shown in SEQ ID NO:3. The primer pair for Streptococcus suis includes a forward primer SS-recNF and a reverse primer SS-recNR, the sequences of which are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively. The probe SS-probe for Streptococcus suis has a fluorescent group FAM at its 5' end and a quencher group BHQ1 at its 3' end, and its nucleotide sequence is shown in SEQ ID NO:6. The concentration of each primer and probe in the kit of the present invention is 10 nmol / L and is stored at -20°C for future use.

[0035] (2) Plasmid standards

[0036] Plasmid standards include Streptococcus pasteurianus plasmid standard and Streptococcus suis plasmid standard.

[0037] Streptococcus pasteurianus plasmid standard: Using the genomic DNA of Streptococcus pasteurianus WUSP067 (genebank accession number NZ_CP039457) as a template, 3230ECOR1-F and 3230XHO1-R as primers, and high-fidelity PCR enzyme to PCR amplify the target fragment. E8M05_ RS03230 (Gene ID: 64018373) was amplified, and the target fragment (sequence shown in SEQ ID NO: 7) was then double-digested with EcoR1 and Xho1 and inserted between the EcoR1 and Xho1 restriction sites of the pet28A vector to construct a recombinant plasmid. After sequencing, a correctly connected Streptococcus pasteurianus recombinant plasmid was finally obtained. The Streptococcus pasteurianus recombinant plasmid was introduced into the competent DH5α strain of Escherichia coli for proliferation. The plasmid was extracted and the sample concentration (unit: ng / μL) was measured using a spectrophotometer. The concentration and copy number conversion formula is: copies / μL = 6.02×10 23 (copies / mol) × plasmid concentration (ng / μL) × 10 -9 / [(vector molecular weight + insert molecular weight) × 660] (g / mol) to obtain the copy number (copies / μL). The recombinant plasmid of Streptococcus pasteurianus was diluted with TE buffer (pH 8.0, 10 mmol / L Tris-HCl buffer containing 1 mmol / L EDTA) to obtain a copy number of 1×10 9 A Streptococcus pasteurianus plasmid standard was prepared at 100 copies / μL. The sequence of 3230ECOR1-F is as follows: 5'-ATGGGTCGCGGATCCGAATTCTTGGAAACGTGTCAGCATGAAG-3'; the sequence of 3230XHO1-R is as follows: 5'-GTGGTGGTGGTGGTGCTCGAGTCACTGCTCATCTCTTAACTGACGA-3'. The sequences of 3230ECOR1-F and 3230XHO1-R are shown in SEQ ID NOs: 26-27, respectively.

[0038] Streptococcus suis plasmid standard: using the genomic DNA of Streptococcus suis P1 / 7 (genebank accession number: NC_012925.1) as a template, recN-ECOR1-F and recN-XHO1-R as primers, and using a high-fidelity PCR enzyme (NoviZan P525) to PCR amplify the target fragment. recN (genebank accession number: AM946016 REGION) was amplified, and the target fragment (sequence shown in SEQ ID NO: 8) was then double-digested with EcoR1 and Xho1 and inserted between the EcoR1 and Xho1 restriction sites of the pet28A vector to obtain a recombinant plasmid. After sequencing, the correctly connected Streptococcus suis recombinant plasmid was finally obtained. The Streptococcus suis recombinant plasmid was introduced into the competent DH5α strain of Escherichia coli for proliferation. The plasmid was extracted and the sample concentration (unit: ng / μL) was measured using a spectrophotometer. The concentration and copy number conversion formula is: copy number (copies / μL) = 6.02×10 23 (copies / mol) × plasmid concentration (ng / μL) × 10 -9 / [(vector molecular weight + insert molecular weight) × 660] (g / mol) to obtain the copy number (copies / μL). The Streptococcus suis recombinant plasmid was diluted with TE buffer (pH 8.0, 10 mmol / L Tris-HCl buffer containing 1 mmol / L EDTA) to obtain a copy number of 1×10 9Streptococcus suis plasmid standard at 100 copies / μL. The sequence of recN-ECOR1-F is as follows: 5'-ATGGGTCGCGGATCCGAATTCTGGCGAATGGGACGCGCC-3'; the sequence of recN-XHO1-R is as follows: 5'-GTGGTGGTGGTGGTGCTCGAGATCCGGATATAGTTCCTCCTTTCA-3'. The sequences of recN-ECOR1-F and recN-XHO1-R are shown in SEQ ID NOs: 28-29, respectively.

[0039] 2. Method for detecting Streptococcus pasteurianus and Streptococcus suis using the kit of the present invention

[0040] The method for detecting Streptococcus pasteurianus and Streptococcus suis using the kit of the present invention comprises the following steps:

[0041] Dual Taqman fluorescence quantitative PCR reactions were performed using total genomic DNA extracted from the test samples as templates, using primer pairs and probes specific for Streptococcus pasteurianus and Streptococcus suis. The reactions were performed on a QuantStudio 6 Flex real-time fluorescence quantitative PCR instrument (qPCR instrument, purchased from ThermoFisher), with the instrument's fluorescence channels set to FAM (channel 1) and VIC (channel 2). Fluorescence signals were collected and the CT values were recorded. The duplex Taqman fluorescence quantitative PCR reaction system, consisting of 25 μL, consisted of the following: 17.45 μL of fluorescent quantitative enzyme reaction solution LTM012 (Qingdao Lijian Biotechnology Co., Ltd.), 0.5 μL of forward primer SP-3230F, 0.5 μL of reverse primer SP-3230R, 0.25 μL of probe SP-probe, 0.4 μL of forward primer SS-recNF, 0.4 μL of reverse primer SS-recNR, 0.5 μL of probe SS-probe, and 5 μL of template. The duplex Taqman fluorescence quantitative PCR amplification protocol was as follows: incubation at 37°C for 2 min; initial denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, followed by annealing and extension at 60°C for 30 s, for a total of 40 cycles. At the same time, enzyme-free ultrapure water (labshark130114010) was used to replace the total genomic DNA extracted from the test sample as a negative control, Streptococcus suis plasmid standard was used to replace the total genomic DNA extracted from the test sample as a positive control for Streptococcus suis, and Streptococcus pasteurianus plasmid standard was used to replace the total genomic DNA extracted from the test sample as a positive control for Streptococcus pasteurianus. Other tests were the same as those for the test samples.

[0042] (2) Determine whether the sample contains Streptococcus pasteurianus and Streptococcus suis based on the fluorescent signal collected in step (1). The specific method is as follows: When the Ct value in channel 1 is ≤35 and there is a specific amplification curve (i.e., a typical S-shaped amplification curve), it is determined to be positive, i.e., the sample contains Streptococcus suis; when the Ct value is greater than 35 or there is no specific amplification curve, the result is determined to be negative, i.e., the sample does not contain Streptococcus suis. When the Ct value in channel 2 is ≤35 and there is a specific amplification curve, it is determined to be positive, i.e., the sample contains Streptococcus pasteurianus; when the Ct value is greater than 35 or there is no specific amplification curve, the result is determined to be negative, i.e., the sample does not contain Streptococcus pasteurianus.

[0043] The negative controls had no Ct values when tested in channels 1 and 2, and the positive controls (Streptococcus pasteurianus and Streptococcus suis plasmid standards) had Ct values ≤ 35 and specific amplification curves when tested in channels 2 and 1.

[0044] Example 2 Performance of the kit of the present invention

[0045] 1. Feasibility verification of the primer and probe combination in the kit of the present invention

[0046] To obtain primers and probes capable of simultaneously detecting S. pasteurianus and S. suis with high sensitivity and specificity, dozens of primer and probe pairs were designed based on the gene sequences of S. pasteurianus and S. suis, and the feasibility of different primer combinations was analyzed to avoid low amplification efficiency and false negatives or false positives caused by primer-probe combinations. During the experiments, it was found that some primers and probes achieved high sensitivity and specificity when used alone to detect S. pasteurianus or S. suis using single-plex qPCR. However, when combined in duplex qPCR to detect both bacteria, there was some interference between the primers and probes, affecting sensitivity and specificity, and even resulting in false negatives or false positives. The target sequence of the universal primer-probe combination for Streptococcus suis in the published patent (primer GDH-F: 5'-GAGCTCTTCTCTACACTTGAGCC-3', primer GDH-R: 5'-CCATGGAACACGGAAGCTG-3', probe GDH-P: 5'-TTGAAGCACACCCAGAATACATCGAAGAA-3'; the nucleotide sequences of primers GDH-F, GDH-R, and probe GDH-P are shown in SEQ ID NOs: 15-17, respectively) was found to be highly homologous to non-target strains through BLAST comparison on the NCBI website, which is prone to false positive results. The results are as follows Figure 1 As shown: This sequence is Streptococcus suivaginalis The similarity was 99.12%, and the primer and probe regions were completely identical. The similarities with two other non-suis Streptococci were 97.37% and 93.52%, respectively, making false positives more likely. Table 1 lists some of the selected sequences.

[0047] Table 1 Single-plex qPCR primers and corresponding Taqman probe sequences for Streptococcus pasteurianus and Streptococcus suis

[0048]

[0049] The three primer-probe compositions for Streptococcus pasteurianus in Table 1 were combined with primer-probe compositions for Streptococcus suis to perform PCR amplification on Streptococcus pasteurianus WUSP067 (BioSample ID: SAMN11431855 ), WUSP068 (BioSample ID: SAMN37358784 ), WUSP069 (BioSample ID: SAMN37358785 ), WUSP070 (BioSample ID: SAMN27738348), ATCC43144 (BioSample ID: SAMD00060984), NCTC13784 (BioSample ID: SAMEA4030747), WUSP074 (BioSample ID: SAMN27738349 ), WUSP082 (BioSample ID: SAMN37357278 ), WUSP083 (BioSample ID: SAMN37358790 ), WUSP084 (BioSample ID: SAMN37358786 ), WUSP085 (BioSample ID: SAMN37358791 ), WUSP086 (BioSample ID: SAMN37358792 ), WUSP087 (BioSample ID: SAMN37358793 ), WUSP088 (BioSample ID: SAMN37358794 ), Streptococcus suis SC070731 (BioSample ID: SAMN02603632), Streptococcus equi subsp. zooepidemicus ATCC35246, Streptococcus agalactiae GD201008-001 (BioSample ID: SAMN02603155 ), Streptococcus aureus ML20171221B6-2, Bacillus subtilis 1.460, Aeromonas hydrophila WUQT018, Klebsiella pneumoniae WUQT019, Streptococcus dysgalactiae WUQT020, Escherichia coli WUQT022, Enterococcus WUQT024, Streptococcus vaginalis WUQT033, Streptococcus pneumoniae ATCC49619, these 26 bacterial genomic DNAs were tested. The specific method is shown in Title 2 of Example 1. The test results are as follows. Figure 2 .Depend on Figure 2It can be seen that the three combinations can simultaneously detect multiple Streptococcus and related zoonotic pathogens, and only Streptococcus pasteurianus and Streptococcus suis are detected normally, while other pathogens are not detected. It proves that there are no false positive results in these three combinations. However, except for Streptococcus pasteurianus primer-probe composition 1, Streptococcus pasteurianus primer-probe composition 2, Streptococcus pasteurianus primer-probe composition 3 and Streptococcus suis primer-probe composition respectively have missed detection phenomena. Among them, the combination of Streptococcus pasteurian primer-probe composition 2 and Streptococcus suis primer-probe composition has a false negative result for WUSP074, while the combination of Streptococcus pasteurian primer-probe composition 3 and Streptococcus suis primer-probe composition can only detect six strains of Streptococcus pasteurianus, namely WUSP067, WUSP068, WUSP069, WUSP070, NCTC13784 and WUSP084, and missed the detection of 8 strains of Streptococcus pasteurianus.

[0050] 2. Reliability of the kit of the present invention

[0051] The reliability of the detection results of the kit of the present invention was demonstrated by establishing a standard curve of the kit of the present invention.

[0052] The Streptococcus pasteurianus plasmid standard was serially diluted with TE buffer (pH 8.0, 10 mmol / L Tris-HCl buffer containing 1 mmol / L EDTA) to obtain concentrations of 1×10 9 copies / μL, 1×10 8 copies / μL, 1×10 7 copies / μL, 1×10 6 copies / μL, 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 Seven concentrations of Streptococcus pasteurianus plasmid standard were prepared. The method for detecting Streptococcus pasteurianus and Streptococcus suis using the kit of the present invention in Example 1 was used. The Streptococcus pasteurianus plasmid standard at each concentration was used as a template for detection. The fluorescence channel of the qPCR instrument was set to VIC. The fluorescence signal was collected by the qPCR instrument, and the CT value was recorded to obtain a fluorescence amplification curve ( Figure 3 A in the figure). A linear regression was performed using the logarithmic value of the concentration of the Streptococcus pasteurianus plasmid standard (logC, where C is the concentration of the Streptococcus pasteurianus plasmid standard and the unit of C is copies / μL) as the abscissa (x) and the corresponding CT value as the ordinate (y) to obtain a standard curve, as shown in the figure. Figure 3 The standard curve for C is as follows: y = -3.3937x + 35.265, R 2 =0.999.

[0053] The Streptococcus suis plasmid standard was serially diluted with TE buffer (pH 8.0, 10 mmol / L Tris-HCl buffer containing 1 mmol / L EDTA) to obtain concentrations of 1×10 9 copies / μL, 1×10 8 copies / μL, 1×10 7 copies / μL, 1×10 6 copies / μL, 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 The seven concentrations of Streptococcus suis plasmid standard were 1000 copies / μL. According to the method of detecting Streptococcus pasteurianus and Streptococcus suis using the kit of the present invention in Example 1, the Streptococcus suis plasmid standard of each concentration was used as a template for detection. The fluorescence channel of the qPCR instrument was set to FAM. At the same time, the fluorescence signal was collected by the qPCR instrument, and the CT value was recorded to obtain the fluorescence amplification curve ( Figure 3 The standard curve was obtained by linear regression using the logarithmic value of the concentration of the Streptococcus suis plasmid standard (logC, C is the concentration of the Streptococcus suis plasmid standard, and the unit of C is copies / μL) as the horizontal axis (x) and the corresponding CT value as the vertical axis (y). Figure 3 The standard curve is as follows: y = -3.4268x + 33.18, R 2 =0.999.

[0054] Depend on Figure 3 It can be seen that the kit of the present invention is used to detect the plasmid standard of Streptococcus pasteurianus and the plasmid standard of Streptococcus suis, and the R 2 All reached 0.999, indicating that the test results of the kit are reliable.

[0055] 3. Sensitivity test

[0056] The kit of the present invention was used to perform the test of the concentration of 1×10 8 copies / μL, 1×10 7 copies / μL, 1×10 6 copies / μL, 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×100 The sensitivity of the detection method was explored by testing the plasmid standards of Streptococcus pasteurianus and Streptococcus suis at nine concentrations of 10 copies / μL. Figure 4 In order to determine the sensitivity of the kit for detecting Streptococcus pasteurianus and Streptococcus suis, the plasmid standards of Streptococcus pasteurianus and Streptococcus suis were serially diluted with TE buffer (pH 8.0, 10 mmol / L Tris-HCl buffer containing 1 mmol / L EDTA). 2 copies / μL, 1×10 1 copies / μL, 1×10 0 The three concentrations of 100 copies / μL were detected using the method described in Example 1, Title 2, with 21 replicates for each concentration. The minimum concentration that achieved a 95% detection rate was taken as the sensitivity of the kit of the present invention. The results are shown in Table 2.

[0057] Table 2 Sensitivity test results of the kit of the present invention for detecting a single pathogen

[0058]

[0059] From Table 2 and Figure 4 It can be seen that the sensitivity of the kit for detecting Streptococcus pasteurianus can reach 1×10 2 copies / μL, Streptococcus suis is 1×10 1 copies / μL.

[0060] 4. Specificity Experiment

[0061] The kit of the present invention is used to detect genomic DNA of 12 bacteria, including Streptococcus pasteurianus WUSP067, Streptococcus suis SC070731, Streptococcus equi subsp. zooepidemicus ATCC35246, Streptococcus agalactiae GD201008-001, Streptococcus multianimalis ML20171221B6-2, Bacillus subtilis 1.460, Aeromonas hydrophila WUQT018, Klebsiella pneumoniae WUQT019, Streptococcus dysgalactiae WUQT020, Escherichia coli WUQT022, Enterococcus WUQT024, and Streptococcus hyovaginosa WUQT033.

[0062] In addition, the kit of the present invention was used to detect the genomic DNA of 15 strains of Streptococcus suis whose serotypes were determined (using serotype-specific PCR), including Streptococcus suis 2021WUSS121 (type 1), 2018WUSS012 (type 1 / 2), 2022WUSS007 (type 2, biosample: SAMN33386794), 2022WUSS148 (type 3, biosample: SAMN38724730), WUSS228 (type 4, biosample: SAMN34237817), and 20 21WUSS081 (Type 5), 2021WUSS006 (Type 7), 2021WUSS036 (Type 8), 2022WUSS046 (Type 9, biosample: SAMN33386820), 2021WUSS017 (Type 12), 2022WUSS024 (Type 14), 2021WUSS001 (Type 16), 2021WUSS015 (Type 21), 2023WUSS018 (Type 24), WUSS416 (Type 31, biosample: SAMN28557614).

[0063] Depend on Figure 5 It can be seen that when the kit of the present invention is used to simultaneously detect multiple Streptococci and related zoonotic diseases, it is found that only Streptococcus pasteurianus and Streptococcus suis are normally detected, while other pathogens are not detected, indicating that the detection method has good specificity.

[0064] Depend on Figure 6 It can be seen that the FAM fluorescence of the kit of the present invention was positive when detecting genomic DNA of 15 strains of Streptococcus suis, and both the positive control and the negative control were established, indicating that the kit of the present invention can specifically identify different serotypes of Streptococcus suis without producing false negatives and has good specificity.

[0065] 5. Repeatability Experiment

[0066] At the same time, the kit of the present invention was used to detect the concentration of 1×10 7 copies / μL, 1×10 5 copies / μL, 1×10 3 Plasmid standards for Streptococcus pasteurianus and Streptococcus suis were tested three times at each concentration, designated as Group 1. The experiment was repeated at two additional times, designated as Groups 2 and 3. The coefficient of variation (CV%) within and between groups was calculated.

[0067] Table 3 Repeatability test results of the kit of the present invention

[0068]

[0069] The stability of the kit of the present invention was determined by comparing the coefficient of variation between and within groups for detecting a single pathogen. As shown in Table 3, most of the coefficients of variation between and within groups were less than 1%, with a few ranging from 1% to 2%, indicating that the kit of the present invention has good stability.

[0070] Example 3 Detection of clinical samples using the kit of the present invention

[0071] (1) Sample collection

[0072] 95 samples of pig tonsils were collected for examination.

[0073] (2) Sample pretreatment

[0074] The porcine tonsils to be examined underwent preliminary processing: rinsed with 1× PBS buffer (pH 7.4) (biosharp BL302A) to remove surface contaminants. The clinical sample surface was wiped with 75% alcohol cotton to reduce bacterial contaminants. The clinical sample was further burned with an alcohol burner. Approximately 0.1 g of the internal sample from the pre-processed clinical sample was placed in a homogenization tube. 900 μL of 1× PBS buffer (pH 7.4) was added and homogenized using an MP FastPrep-24 homogenizer (USA). The homogenate was transferred to 5 mL of THB liquid medium at a ratio of 1:50. Polymyxin and nalidixic acid were added to the THB liquid medium to a concentration of 15 mg / L and 30 mg / L, respectively. The culture was incubated at 37°C, 5% CO₂ for 8–10 h.

[0075] (3) Extraction of total genomic DNA

[0076] Take 2 mL of the liquid culture obtained in step (2) and use DNA / RNA Extraction Kit (Prepackaged) RM201 (Nanjing Novezan Biotechnology Co., Ltd.) according to the product instructions to extract the total genomic DNA of the sample and store it at -20℃ for later use.

[0077] (4) Conventional PCR testing

[0078] According to the published multiplex PCR method of Streptococcus pasteurianus (Ma M, Wang S, Zhu X, et al. The Identification of Streptococcus pasteurianusObtained from Six Regions in China by Multiplex PCR Assay and the Characteristics of Pathogenicity andAntimicrobial Resistance of This Zoonotic Pathogen[J].Pathogens, 2023, 12(4):12.DOI:10.3390 / pathogens12040615.) and the conventional PCR method for Streptococcus suis were used to perform conventional PCR on the total genomic DNA of the sample obtained in step (3), and then the PCR amplification products were detected by 1.5% agarose gel electrophoresis.

[0079] Among them, the primers used in the multiplex PCR method for Streptococcus pasteurianus include:

[0080] 1-F:5'-GTAGATACTGATGGAGATGGT-3'; 1-R:5'-ATAATCGCCTGGTTGAGTC-3';

[0081] 2-F:5'-TTGTTCCGTTGTCAGCATA-3'; 2-R:5'-AGCACCGATTCTATCCATAA-3';

[0082] 3-F: 5'-GTTCTGGAATGGTTAGGAATC-3'; 3-R: 5'-AAGCAGCGCAATATCAA-3'.

[0083] The sequences of primers 1-F, 1-R, 2-F, 2-R, 3-F and 3-R are shown in SEQ ID NOs: 18-23, respectively.

[0084] Primers used in common PCR methods for Streptococcus suis include:

[0085] recN-F: 5'-CTACAAACAGCTCTCTTCT-3'; recN-R: 5'-ACAACAGCCAATTCATGGCGTGATT-3'.

[0086] The sequences of primers recN-F and recN-R are shown in SEQ ID NOs: 24-25, respectively.

[0087] The standard PCR method for Streptococcus suis uses a 25 μL PCR reaction system composed of 12.5 μL of Quick Taq HSDyeMix (TOYOBO), 1 μL of the forward primer recN-F, 1 μL of the reverse primer recN-R, 8.5 μL of ddH2O, and 2 μL of the template. The PCR program is as follows: 94°C for 2 min; 30 cycles of 94°C for 30 s, 60°C for 15 s, and 68°C for 30 s; and 68°C for 5 min.

[0088] (5) The kit of the present invention performs dual fluorescence quantitative PCR detection

[0089] The total genomic DNA of the sample obtained in step (3) was detected using the kit and detection method of the present invention in Example 1.

[0090] (6) Detection results of Streptococcus pasteurianus and Streptococcus suis in pig tonsils

[0091] The results of conventional PCR detection and detection by the kit of the present invention are shown in Table 4.

[0092] Table 4 Detection results of each method

[0093]

[0094] As shown in Table 4, the kit of the present invention detected 5 more positive samples for Streptococcus pasteurianus and 14 more for Streptococcus suis than the conventional PCR method. These results demonstrate that the kit of the present invention significantly outperforms the previously published conventional PCR method, reducing the occurrence of false negatives and enabling the detection of mixed infections in a single test. This eliminates the need for secondary PCR validation and subsequent PCR amplification processing, providing a new method for the rapid and accurate detection of Streptococcus pasteurianus and Streptococcus suis in samples.

Claims

1. A dual fluorescence quantitative PCR kit for simultaneous detection of Streptococcus pasteurianus and Streptococcus suis, comprising a primer pair for Streptococcus pasteurianus, a probe SP-probe for Streptococcus pasteurianus, a primer pair for Streptococcus suis, and a probe SS-probe for Streptococcus suis; the primer pair for Streptococcus pasteurianus comprises a forward primer SP-3230F and a reverse primer SP-3230R, the sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the probe SP-probe for Streptococcus pasteurianus has a fluorescent group VIC at its 5' end and a quenching group BHQ1 at its 3' end, the nucleotide sequence of which is shown in SEQ ID NO: 3; the primer pair for Streptococcus suis comprises a forward primer SS-recNF and a reverse primer SS-recNR, the sequences of which are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively. NO:5 shows; the probe SS-probe for Streptococcus suis has a fluorescent group FAM at the 5' end and a quenching group BHQ1 at the 3' end, and its nucleotide sequence is shown in SEQ ID NO:

6.

2. The kit according to claim 1, wherein The concentration of each primer and probe in the kit is 8-12 nmol / L.

3. The kit according to claim 1 or 2, characterized in that The kit also includes a Streptococcus pasteurianus plasmid standard and a Streptococcus suis plasmid standard. The Streptococcus pasteurianus plasmid standard and the Streptococcus suis plasmid standard are respectively E8M05_RS03230 Gene fragments and Streptococcus suis recN The gene fragment is inserted into the vector.

4. The kit according to claim 3, wherein The concentrations of the Streptococcus pasteurianus plasmid standard and the Streptococcus suis plasmid standard were both 1×10 9 copies / μL-1×10 2 copies / μL.

5. A method for detecting Streptococcus pasteurianus and Streptococcus suis using the kit according to claim 1 for non-diagnostic purposes, comprising the following steps: Dual Taqman fluorescence quantitative PCR reactions were performed using total genomic DNA extracted from the test sample as a template using primer pairs and probes targeting Streptococcus pasteurianus and Streptococcus suis. The fluorescence channels of the qPCR instrument were set as follows: Channel 1: FAM, Channel 2: VIC. When the Ct value in Channel 1 was ≤35 and a specific amplification curve was present, the result was considered positive, indicating that the sample contained Streptococcus suis. When the Ct value in Channel 2 was ≤35 and a specific amplification curve was present, the result was considered positive, indicating that the sample contained Streptococcus pasteurianus.

6. The method according to claim 5, characterized in that The dual Taqman fluorescence quantitative PCR reaction amplification procedure was as follows: incubation at 37°C for 2 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, for a total of 40 cycles.

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