Primer probe combination, kit and detection method for quantitatively detecting group B streptococcus nucleic acid

Abstract

The invention belongs to the field of molecular diagnostic biology, and relates to a primer probe combination, a kit and a detection method for quantitatively detecting group B streptococcus nucleic acid, and the primer probe combination comprises an upstream primer for detecting group B streptococcus and has a nucleotide sequence as shown in SEQ ID NO: 1; the downstream primer for detecting the group B streptococcus has a nucleotide sequence as shown in SEQ ID NO: 2; the group B streptococcus detection probe has a nucleotide sequence as shown in SEQ ID NO: 3; the internal control gene upstream primer has a nucleotide sequence as shown in SEQ ID NO: 4; the internal control gene downstream primer has a nucleotide sequence as shown in SEQ ID NO: 5; and the internal control gene probe has a nucleotide sequence as shown in SEQ ID NO: 6. The method is simple to operate and high in detection efficiency, and can be used for qualitative and quantitative detection of group B streptococcus at the same time, and the primer probe combination is high in characteristic and high in detection sensitivity and detection accuracy of group B streptococcus.

Description

Technical Field

[0001] This application relates to the technical field of molecular diagnostic biology, and particularly to a primer-probe combination, a kit and a detection method for quantitatively detecting the nucleic acid of group B streptococcus. Background Art

[0002] Group B Streptococcus (GBS) is a β-hemolytic Gram-positive bacterium that is usually colonized in the gastrointestinal tract or lower genital tract of women without obvious clinical symptoms, but is an invasive pathogen in other hosts. GBS rarely causes maternal sepsis, but is likely to cause fetal stillbirth and premature birth, and also causes neonatal GBS infection.

[0003] GBS is the main cause of the incidence and mortality of neonatal infections globally. After fetal infection with GBS, the most common clinical manifestations are sepsis, pneumonia, meningitis and encephalopathy. Research shows that GBS is the main pathogenic bacterium causing neonatal meningitis and sepsis, with a global incidence rate of about 0.53‰ and a mortality rate as high as 10%. Even after complete recovery, nearly 30% of the surviving children will retain long-term neurological sequelae such as deafness, visual impairment, developmental disorders and cerebral palsy.

[0004] Currently, the detection methods related to the detection of group B streptococcus include bacterial isolation and culture method, colloidal gold immunochromatography method and fluorescence quantitative PCR detection method, etc. Among them, the bacterial culture method has a long detection time, high cost, low detection sensitivity and low positive detection rate; the mass spectrometry rapid identification method has relatively low detection sensitivity and a relatively long time (about 6h), and when there are two or more bacteria in the culture solution, it is often impossible to accurately identify the target strain; the colloidal gold immunochromatography method is prone to false positives and is not suitable for directional diagnosis; real-time fluorescence PCR has the advantages of high sensitivity, high accuracy and rapid detection, and is used for perinatal screening and rapid detection in emergency situations, but its quantification requires reference standards, standard curves and internal standard calibration fluorescence, and it has poor resolution for low-copy target gene molecules, and the sensitivity, precision and resolution are limited. Summary of the Invention

[0005] The purpose of this application is to provide a primer-probe combination, a kit and a detection method for quantitatively detecting the nucleic acid of group B streptococcus, so as to solve the technical problems in the prior art that the nucleic acid of group B streptococcus cannot be quantitatively detected quickly and accurately, and the detection sensitivity, precision and resolution are relatively low due to the dependence on the standard curve.

[0006] In order to solve the above technical problems, the embodiment of this application provides a primer-probe combination for quantitatively detecting the nucleic acid of group B streptococcus, and adopts the following technical scheme:

[0007] Comprising a Streptococcus agalactiae upstream primer with a nucleotide sequence as shown in SEQ ID NO: 1;

[0008] A Streptococcus agalactiae downstream primer with a nucleotide sequence as shown in SEQ ID NO: 2;

[0009] A Streptococcus agalactiae probe with a nucleotide sequence as shown in SEQ ID NO: 3;

[0010] An internal control gene upstream primer with a nucleotide sequence as shown in SEQ ID NO: 4;

[0011] An internal control gene downstream primer with a nucleotide sequence as shown in SEQ ID NO: 5;

[0012] An internal control gene probe with a nucleotide sequence as shown in SEQ ID NO: 6.

[0013] Furthermore, the 5'-end of the probe is labeled with a fluorescent group, and the 3'-end of the probe is labeled with a quenching group;

[0014] wherein, the fluorescent group is selected from FAM, VIC or HEX; the quenching group is selected from MGB, BHQ series, TAMRA or Eclipse.

[0015] Furthermore, the 5'-end of the Streptococcus agalactiae probe is labeled with a FAM fluorescent group, and the 3'-end of the Streptococcus agalactiae probe is labeled with an MGB quenching group; the 5'-end of the internal control gene probe is labeled with a VIC fluorescent group, and the 3'-end of the internal control gene probe is labeled with an MGB quenching group.

[0016] To solve the above technical problems, the embodiments of the present application further provide a kit for quantitatively detecting Streptococcus agalactiae nucleic acid, adopting the following technical solutions:

[0017] Comprising a primer-probe mixture, a ddPCR premix, a GBS positive control product and a GBS negative control product; the primer-probe mixture comprises the primer-probe combination as described above.

[0018] Furthermore, the final concentration of the Streptococcus agalactiae upstream primer and the Streptococcus agalactiae downstream primer in the PCR reaction system is 0.5 μmol / L; the final concentration of the Streptococcus agalactiae probe in the PCR reaction system is 0.25 μmol / L; the final concentration of the internal control gene upstream primer and the internal control gene downstream primer in the PCR reaction system is 0.5 μmol / L; the final concentration of the internal control gene probe in the PCR reaction system is 0.25 μmol / L.

[0019] Further, the components of the GBS positive control product include a Streptococcus group B pseudovirus with a concentration of not less than 1×10 5 copies / mL and a pseudovirus containing an internal standard fragment with a concentration of not less than 1×10 5 copies / mL; the components of the GBS negative control product include a pseudovirus containing an internal standard fragment with a concentration of not less than 5×10 4 copies / mL and TE buffer.

[0020] Further, the test sample of the kit is human sputum;

[0021] The criteria for the kit to determine a valid test include:

[0022] In each test, only the reaction wells with a droplet count ≥ 10,000 in all reaction wells are valid reaction wells;

[0023] Among the valid reaction wells, there should be a negative control group and a positive control group. When the test result of the positive control group is positive, the test result of the negative control group is negative, and the concentration test result of the positive control group is 105 ± 10% copies / mL, the test result is valid.

[0024] To solve the above technical problems, the embodiment of the present application also provides a method for quantitatively detecting Streptococcus group B nucleic acid for non-diagnostic purposes. The detection is performed using the above-mentioned kit, and the following technical solutions are adopted:

[0025] Collect the sample to be tested, extract nucleic acid from the sample to be tested to obtain the nucleic acid of the sample to be tested;

[0026] Take out the primer-probe mixture and ddPCR premix from the kit, melt them at room temperature and mix well, prepare the PCR reaction system, and aliquot the PCR reaction system into a preset number of PCR reaction tubes;

[0027] Add the nucleic acid of the sample to be tested, GBS positive control product, and GBS negative control product to the PCR reaction tubes respectively, perform instantaneous centrifugation and then transfer them to the droplet preparation area;

[0028] Perform droplet preparation on the liquid in the PCR reaction tubes, and perform PCR amplification reaction on the prepared droplets according to the preset PCR reaction conditions to obtain the PCR reaction product;

[0029] Perform quantitative analysis on the PCR reaction product to obtain the copy number of Streptococcus group B nucleic acid.

[0030] Further, the PCR reaction conditions are as follows:

[0031] Incubate at 95°C for 10 min for hot start of the enzyme; denature at 94°C for 30 s, anneal at 58°C for 1 min, for a total of 45 cycles; finally, inactivate the enzyme at 98°C for 10 min.

[0032] Furthermore, the detection method further includes:

[0033] Dilute the Streptococcus agalactiae reference products S1 - S3 with RNase - free deionized water at ratios of 1:10, 1:100, 1:1000, 1:10000, and 1:100000 respectively.

[0034] Compared with the prior art, the present application mainly has the following beneficial effects:

[0035] The primer - probe combination, kit, and detection method for quantitatively detecting Streptococcus agalactiae nucleic acid provided by the present application are based on droplet digital PCR technology, quantitatively detect the Streptococcus agalactiae nucleic acid in the sample, and can obtain the quantitative detection result in as fast as 3 h. Samples with a concentration as low as 3500 copies / mL can be stably detected. The operation is simple, the detection efficiency is high, and it can simultaneously perform qualitative and quantitative analysis on Streptococcus agalactiae. Secondly, the primer - probe combination of the present application has high specificity, high detection sensitivity and high detection accuracy for Streptococcus agalactiae. In addition, the present application does not require setting a standard curve. According to the results of fluorescence type and the number of fluorescent droplets, the positive and negative of Streptococcus agalactiae and the copy number can be directly judged, greatly simplifying the operation steps. Brief Description of the Drawings

[0036] In order to more clearly illustrate the solutions in the present application, the following will briefly introduce the drawings required for the description of the embodiments of the present application. Obviously, the following - described drawings are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0037] Figure 1 It is a schematic diagram of the PCR reaction result of the kit of the present application for detecting the GBS negative control product;

[0038] Figure 2 It is a schematic diagram of the PCR reaction result of the kit of the present application for detecting the GBS positive control product;

[0039] Figures 3 to 7 They are successively the PCR reaction results of the kit of the present application for detecting the Streptococcus agalactiae enterprise reference product S1 diluted 1:10, diluted 1:100, diluted 1:1000, diluted 1:10000, and diluted 1:100000;

[0040] Figures 8 to 12PCR reaction results of the kit of the present application for detecting Group B streptococcus enterprise reference product S2 diluted 1:10, 1:100, 1:1000, 1:10000, and 1:100000 in sequence;

[0041] Figures 13 to 17 PCR reaction results of the kit of the present application for detecting Group B streptococcus enterprise reference product S3 diluted 1:10, 1:100, 1:1000, 1:10000, and 1:100000 in sequence;

[0042] Figure 18 are the PCR reaction results of the kit of the present application for detecting Group B streptococcus enterprise reference products PC01 - PC05;

[0043] Figure 19 are the PCR reaction results of the kit of the present application for detecting Group B streptococcus enterprise reference products NC01 - NC10;

[0044] Figure 20 are the detection results of the clinical samples by the kit of the present application and a third - party kit. Detailed implementation manners

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs; the terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above - mentioned drawings are intended to cover non - exclusive inclusion. The terms "first", "second", etc. in the specification and claims of this application or the above - mentioned drawings are used to distinguish different objects and are not used to describe a specific order.

[0046] Reference to "embodiment" herein means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of this application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0047] To enable those skilled in the art to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0048] The primer-probe combination, kit and detection method for quantitative detection of group B streptococcus nucleic acid provided in the present application adopt droplet digital PCR (hereinafter referred to as "ddPCR") technology to quantitatively detect group B streptococcus in extracted sputum, and have the advantages of simple operation, simultaneous qualitative and quantitative detection, and high detection efficiency.

[0049] Based on the droplet digital PCR technology, the present application proposes a primer-probe combination for quantitative detection of group B streptococcal nucleic acid, the primer-probe combination comprising:

[0050] An upstream primer for detecting group B streptococci (GBS upstream primer) has a nucleotide sequence as shown in SEQ ID NO: 1; a downstream primer for detecting group B streptococci (GBS downstream primer) has a nucleotide sequence as shown in SEQ ID NO: 2; a probe for detecting group B streptococci (GBS detection probe) has a nucleotide sequence as shown in SEQ ID NO: 3;

[0051] The upstream primer of the internal control gene has the nucleotide sequence shown in SEQ ID NO:4; the downstream primer of the internal control gene has the nucleotide sequence shown in SEQ ID NO:5; and the internal control gene probe has the nucleotide sequence shown in SEQ ID NO:6.

[0052] In this application, the design of primers and probes shall give priority to following the general design principles of primers and probes, that is, the GC% of primers and probes is required to be between 30% and 70%, the theoretical Tm>48°C, and the Tm value of the probe is usually 5-10°C higher than the Tm value of the primers; the 3' ends of the primers and probes themselves, and between the primers and probes, should avoid base pairing as much as possible; at the same time, the 3' ends of the primers should avoid 3 or more consecutive identical bases, and the probes should avoid 4 consecutive Gs, or 6 consecutive A's, to avoid mismatch problems. At the same time, the principle of specific design must be followed. The selected target gene primers and probes are required to be designed in the conserved region of the target gene, which is specific only to the target gene and has no cross-reaction with other species and human genes. The probe is located in the area between the primers. Primers were designed according to the above principles, and a large number of experiments and screening were carried out on the designed specific primers and probes, which were then combined, optimized and verified. By exploring the reaction conditions and optimizing the methods, the linearity, specificity, sensitivity and repeatability of different primers and probes were compared. Finally, the optimal primer-probe combination with no mutual interference after combination, high amplification efficiency and good specificity was selected, as shown in Table 1.

[0053] Table 1 Group B Streptococcus CAMP gene detection site and internal control primer probe nucleotide sequence table

[0054]

[0055]

[0056] This application designs primers for the human CAMP gene detection site, and designs a pair of specific PCR primers (i.e., the upstream and downstream primers for detecting group B streptococcus) and a hybridization probe (i.e., the probe for detecting group B streptococcus) for this detection site, which are used to hybridize the amplification products of the CAMP gene detection site. At the same time, a pair of amplification primers (i.e., the upstream and downstream primers for the internal control gene) and a hybridization probe (i.e., the internal control probe) are designed according to the sequence of the internal control gene. Among them, the internal control gene can be selected as the glyceraldehyde-3-phosphate dehydrogenase gene fragment, that is, the GAPDH amplification fragment.

[0057] The 5' end of the above probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. Among them, the fluorescent group is selected from FAM, VIC or HEX; the quenching group is selected from MGB, BHQ series, TAMRA or Eclipse.

[0058] The fluorescent groups labeled at the 5' ends of the probe for detecting group B streptococcus and the internal control gene probe are different, so as to obtain the detection results according to different fluorescence channels. Among them, the 5' end of the probe for detecting group B streptococcus is labeled with a FAM fluorescent group, and the 3' end of the probe for detecting group B streptococcus is labeled with an MGB quenching group; the 5' end of the internal control gene probe is labeled with a VIC fluorescent group, and the 3' end of the internal control gene probe is labeled with an MGB quenching group.

[0059] The primers for detecting group B streptococcus and the probe for detecting group B streptococcus labeled with the FAM fluorescent group can be used for the quantitative detection of group B streptococcus. According to the presence or absence of the number of positive droplets in the FAM channel and the number of positive and negative droplets, it can be judged whether the sample contains group B streptococcus and calculate the concentration of the target fragment of group B streptococcus in the sample.

[0060] This application optimizes the specificity of the primer and probe combination for detecting group B streptococcus, reduces the misdiagnosis probability caused by non-specific binding, and designs an internal control gene detection system for monitoring and correction.

[0061] This application also provides a kit for quantitatively detecting group B streptococcus nucleic acid, which includes a primer-probe mixture, a ddPCR premix, a GBS positive control product and a GBS negative control product; the primer-probe mixture includes the primer-probe combination as described above.

[0062] Among them, the final concentration of the upstream primer for detecting group B streptococcus and the downstream primer for detecting group B streptococcus in the PCR reaction system is 0.5 μmol / L; the final concentration of the probe for detecting group B streptococcus in the PCR reaction system is 0.25 μmol / L; the final concentration of the upstream primer for the internal control gene and the downstream primer for the internal control gene in the PCR reaction system is 0.5 μmol / L; the final concentration of the probe for the internal control gene in the PCR reaction system is 0.25 μmol / L.

[0063] The ddPCR premix can be the ddPCR Supermix for Probes (No dUTP) for RNA samples provided by Bio-rad, with the product numbers: 1863023, 1863024 or 1863025.

[0064] This application also includes a positive control group and a negative control group. The positive control group is a GBS positive control product. The components of the GBS positive control product include group B streptococcus pseudovirus with a concentration of not less than 1×10 5 copies / mL and pseudovirus containing an internal standard fragment with a concentration of not less than 1×10 5 copies / mL; the components of the GBS negative control product include pseudovirus containing an internal standard fragment with a concentration of not less than 5×10 4 copies / mL and TE buffer.

[0065] The detection sample applicable to the kit of this application is human sputum.

[0066] The criteria for the kit of this application to determine the detection effectiveness are:

[0067] Only the reaction wells with a droplet count ≥ 10,000 in all reaction wells for each detection are valid reaction wells;

[0068] Among the valid reaction wells, the negative control group and the positive control group should be included. When the detection result of the positive control group is positive, the detection result of the negative control group is negative, and the concentration detection result of the positive control group is 10 5 ±10% copies / mL, the detection result is valid.

[0069] Based on the above kit for quantitatively detecting group B streptococcus nucleic acid, this application also provides a detection method for quantitatively detecting group B streptococcus nucleic acid for non-diagnostic purposes using the kit as described above, including the following steps:

[0070] Step S10, collect the sample to be tested, extract nucleic acid from the sample to be tested to obtain the nucleic acid of the sample to be tested.

[0071] The sample to be tested is a human sputum sample. Among them, before collecting the sputum sample, the patient needs to perform thorough oral cleaning, cough deeply forcefully to cough up the sputum, and collect it into a sterile cup for subsequent testing.

[0072] After the sample collection is completed, a qualified human sputum extraction kit is used to extract the sampling solution. For example, a nucleic acid extraction or purification reagent (registration number: Yue Sui Xie Bei 20170583) produced by Da'an Gene Co., Ltd. is used to extract and purify the nucleic acid from the collected sputum sample to obtain the nucleic acid of the sample to be tested.

[0073] Step S20: Take out the primer-probe mixture and ddPCR premix from the kit, melt them at room temperature and mix well, prepare the PCR reaction system, and dispense the PCR reaction system into a preset number of PCR reaction tubes.

[0074] Take out the primer-probe mixture and ddPCR premix from the kit, melt them at room temperature and mix well with a vortex oscillator. After centrifuging at 8000 rpm - 10000 rpm for 10 - 20 s, prepare the PCR reaction system, and dispense the PCR reaction system into a preset number of PCR reaction tubes.

[0075] Among them, the preset number N = the number of samples to be tested + GBS negative control + GBS positive control. The single-person PCR reaction system is shown in Table 2.

[0076] Table 2 Single-person PCR reaction system

[0077] Component Volume Primer-probe mixture 5 μL ddPCR premix 10 μL

[0078] Step S30: Add the nucleic acid of the sample to be tested, GBS positive control, and GBS negative control to the PCR reaction tubes respectively, perform instantaneous centrifugation and then transfer them to the droplet preparation area.

[0079] Taking the above single-person PCR reaction system as an example, add 5 μL of the nucleic acid of the sample to be tested, GBS positive control, and GBS negative control to the PCR reaction tubes after adding the PCR reaction system respectively, tighten the tube caps, perform instantaneous centrifugation for 15 seconds and then transfer them to the droplet preparation area.

[0080] Step S40: Prepare droplets from the liquid in the PCR reaction tubes, and perform a PCR amplification reaction on the prepared droplets according to the preset PCR reaction conditions to obtain the PCR reaction product.

[0081] Prepare droplets according to the instruction manual of the Bio-rad droplet digital PCR platform, and transfer the prepared droplets to the Bio-rad special 96-well plate for droplet digital PCR.

[0082] Specifically, the PCR reaction system with added samples was mixed evenly using a vortex oscillator. Take out a droplet preparation chip, transfer a column (8) of reaction systems to the sample wells of the chip, add 70 μL of droplet preparation oil to the droplet preparation oil wells, and send it into the droplet preparation instrument for droplet preparation. After the instrument reports that the droplet preparation is completed, carefully transfer the prepared droplets to a dedicated 96-well plate using a pipette, discard the droplet preparation chip, and then take another droplet preparation chip to process the next column of reaction systems until all the reaction systems to be tested are processed. Then seal the 96-well plate using an aluminum mold on a heat sealer.

[0083] Transfer the sealed 96-well plate to a qualitative PCR, and perform a PCR amplification reaction on the prepared droplets according to the PCR reaction conditions set in Table 3. Among them, the PCR reaction conditions are: enzyme heat activation at 95 °C for 10 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 1 min, for a total of 45 cycles; finally, enzyme inactivation at 98 °C for 10 min.

[0084] Table 3 PCR reaction conditions

[0085]

[0086] After the PCR reaction is completed, take out the 96-well plate containing the PCR reaction product and transfer it to a droplet reader.

[0087] Step S50, perform quantitative analysis on the PCR reaction product to obtain the copy number of Streptococcus agalactiae nucleic acid.

[0088] Set the droplet reading parameters according to the instructions. Note the fluorescence channel selection: select the FAM channel to detect Streptococcus agalactiae nucleic acid and select the VIC channel to detect the internal standard (internal control gene). After setting, start the droplet reading program.

[0089] After the droplet reading is completed, the results are automatically saved and enter the result analysis interface. Check the total number of droplets in each reaction well. If the number of droplets in a certain reaction well < 10000, it is regarded as an invalid reaction well. Then check whether the positive and negative quality control products of this test are included in the valid reaction wells. If they are included, this test is valid.

[0090] If the test is valid, check the fluorescence scatter plot of Ch1 (FAM channel). After drawing the threshold line, check the concentration of Ch1, in units of copies / μL. Finally, the concentration of the original sample can be calculated in combination with the sample dilution factor.

[0091] The detection principle of this application is as follows:

[0092] This kit uses Bio-rad's droplet digital PCR technology to design specific primers and probes in the conserved region of the Streptococcus agalactiae genome. Streptococcus agalactiae is labeled with FAM fluorescence, and the internal standard gene is labeled with VIC fluorescence. After preparing the PCR reaction system and loading the samples, they are dispersed into tens of thousands of droplets. Each droplet can perform PCR reactions simultaneously. After the PCR reactions are completed, a droplet reader is used to detect the fluorescence levels of each droplet to determine the negativity or positivity of the droplets. After counting the negativity and positivity of all droplets, it can be determined whether Streptococcus agalactiae nucleic acid exists in the sample. Further, based on the statistical results of the negative and positive droplets and combined with statistical formulas, the copy number of Streptococcus agalactiae nucleic acid in the sample can be calculated.

[0093] The following will further illustrate the content of this application with specific examples and elaborate on this application, but these examples are by no means restrictive of this application.

[0094] Example 1

[0095] This example provides the composition, packaging, and quantity (96 person-times / box) of a kit for quantitatively detecting Streptococcus agalactiae nucleic acid, as shown in Table 4.

[0096] Table 4 Composition, packaging, and quantity of a kit for quantitatively detecting Streptococcus agalactiae nucleic acid

[0097]

[0098] Using the kit of this example to detect GBS negative control products and GBS positive control products, the schematic diagram of the detection results is shown in Figure 1 and Figure 2 . Among them, Ch1 corresponds to the FAM fluorescence channel for detecting Streptococcus agalactiae gene fragments, and Ch2 corresponds to the VIC fluorescence channel for detecting internal standard fragments. Figure 1 In, no signal is detected in the FAM fluorescence channel, and a fluorescence signal is detected in the VIC fluorescence channel; Figure 2 In, fluorescence signals can be detected in both the FAM fluorescence channel and the VIC fluorescence channel.

[0099] Example 2

[0100] This example conducts a detection experiment on the sample detection range and sensitivity of the kit of this application. The specific process is as follows:

[0101] Take an appropriate number of copies of the GBS primer-probe mixture and ddPCR Supermix for Probes (No dUTP). After preparing the PCR reaction system in the manner of step S20, transfer the PCR reaction system into a PCR reaction tube. Detect the Group B streptococcus enterprise reference products S1, S2, and S3, dilute them with RNase-free deionized water at ratios of 1:10, 1:100, 1:1000, 1:10000, and 1:100000. After nucleic acid extraction, take 5 μL of the extraction solution and add it to the eight-well tube of the PCR reaction system prepared in step 20 to make the total volume 20 μL. Tighten the lids of the eight tubes, mix them vigorously with an oscillator for 15 seconds, centrifuge instantaneously for 15 seconds, and then transfer them to the droplet preparation area.

[0102] After preparing the droplets according to the droplet preparation method of step S40 above, seal the 96-well plate with an aluminum mold on a heat sealer. Transfer the sealed 96-well plate to a qualitative PCR, and perform a PCR reaction on the prepared droplets according to the PCR reaction conditions in Table 3. After the reaction ends, the reaction results are shown in Table 5 and Figures 3 to 17 , Figures 3 to 17 are the schematic diagrams of the PCR reaction results of the Group B streptococcus enterprise reference products S1, S2, and S3 diluted at ratios of 1:10, 1:100, 1:1000, 1:10000, and 1:100000 in sequence.

[0103] Table 5 Detection Range and Sensitivity Detection Results of Samples

[0104]

[0105]

[0106] It can be seen from the detection results in the above table that when the sample concentration is between 3500 and 7500000 copies / mL, the quantitative test results of the kit in this application have a difference of <15% from the true concentration. The results show that the minimum detection limit of the kit in this application is 3500 copies / mL, and the kit has high sensitivity.

[0107] Example Three

[0108] In this example, the accuracy of the kit in this application is detected. Specifically, detect the Group B streptococcus enterprise reference products PC01 - PC05, NC01 - NC10. Take 5 μL of the extraction solution and add it to the eight-well tube of the PCR reaction system prepared in step 20 to make the total volume 20 μL. Tighten the lids of the eight tubes, mix them vigorously with an oscillator for 15 seconds, centrifuge instantaneously for 15 seconds, and then transfer them to the droplet preparation area.

[0109] After the droplets are prepared by the droplet preparation method of step S40 above, seal the 96-well plate with an aluminum mold on a heat sealer, transfer the sealed 96-well plate to a qualitative PCR, and perform a PCR reaction on the prepared droplets according to the PCR reaction conditions in Table 3. After the reaction is completed, the reaction results are shown in Table 6 and Figure 18 and Figure 19 as shown

[0110] Table 6 Accuracy test results

[0111]

[0112]

[0113] According to the results shown in the above table, the positive and negative coincidence rate of the accuracy test of each reference product is 100%, indicating that the accuracy test of the kit of the present invention meets the requirements, and further indicating that the detection accuracy of the kit of the present application is high and the quantitative results are accurate.

[0114] Example 4

[0115] This example is a clinical application experiment, using the kit of the present application and a third-party influenza A detection kit to detect the positive and negative of the tested clinical samples.

[0116] Select 10 clinical positive samples and 10 negative samples of group B streptococcus respectively. After nucleic acid extraction, mark the samples well and ensure that the label information is correct, and store them at -80 °C. During the experiment, take 5 μL of each sample and add it to a reaction tube containing the PCR reaction solution prepared in step one to make the total volume 20 μL. Vigorously shake and mix for 15 seconds with an oscillator, transfer it to the droplet preparation area after instantaneous centrifugation for 15 seconds.

[0117] After the droplets are prepared by the droplet preparation method of step S40 above, seal the 96-well plate with an aluminum mold on a heat sealer, transfer the sealed 96-well plate to a qualitative PCR, and perform a PCR reaction on the prepared droplets according to the PCR reaction conditions in Table 3. After the reaction is completed, the reaction results are shown in Table 7 and Figure 20 as shown

[0118] Table 7 Clinical sample test results of the kit of the present application and the third-party kit

[0119]

[0120]

[0121] As can be seen from the test results in the above table, among the 20 samples, 10 are Streptococcus B samples and 10 are negative samples. The consistency between the test results and those of the third-party kit is 100%. By using the kit of the present application, it is possible to identify whether the tested population carries Streptococcus B and perform quantitative detection on it, which is worthy of popularization and application.

[0122] Obviously, the embodiments described above are only a part of the embodiments of the present application, rather than all of them. The accompanying drawings show the preferred embodiments of the present application, but do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or perform equivalent replacements on some of the technical features. Any equivalent structure made by using the specification and drawings of the present application, directly or indirectly applied in other related technical fields, shall be within the scope of the patent protection of the present application by the same token.

Claims

1. A primer-probe combination for quantitatively detecting the nucleic acid of group B streptococcus, characterized in that, Comprising: An upstream primer for detecting Streptococcus agalactiae, having the nucleotide sequence shown in SEQ ID NO: 1; A downstream primer for detecting Streptococcus agalactiae, having the nucleotide sequence shown in SEQ ID NO: 2; A probe for detecting Streptococcus agalactiae, having the nucleotide sequence shown in SEQ ID NO: 3; An upstream primer for an internal control gene, having the nucleotide sequence shown in SEQ ID NO: 4; A downstream primer for an internal control gene, having the nucleotide sequence shown in SEQ ID NO: 5; A probe for an internal control gene, having the nucleotide sequence shown in SEQ ID NO:

6.

2. The primer-probe combination for quantitatively detecting group B streptococcus nucleic acid according to claim 1, wherein The 5'-end of the probe is labeled with a fluorescent group, and the 3'-end of the probe is labeled with a quenching group; Wherein, the fluorescent group is selected from FAM, VIC or HEX; the quenching group is selected from MGB, BHQ series, TAMRA or Eclipse.

3. The primer-probe combination for quantitatively detecting group B streptococcus nucleic acid according to claim 1, wherein The 5'-end of the probe for detecting Streptococcus agalactiae is labeled with a FAM fluorescent group, and the 3'-end of the probe for detecting Streptococcus agalactiae is labeled with an MGB quenching group; the 5'-end of the probe for the internal control gene is labeled with a VIC fluorescent group, and the 3'-end of the probe for the internal control gene is labeled with an MGB quenching group.

4. A kit for quantitatively detecting the nucleic acid of group B streptococcus, characterized in that, Comprising a primer-probe mixture, a ddPCR premix, a GBS positive control product and a GBS negative control product; the primer-probe mixture comprises the primer-probe combination according to any one of claims 1 to 3.

5. The kit for quantitatively detecting group B streptococcus nucleic acid according to claim 4, wherein The final concentration of the upstream primer for detecting Streptococcus agalactiae and the downstream primer for detecting Streptococcus agalactiae in the PCR reaction system is 0.5 μmol / L; the final concentration of the probe for detecting Streptococcus agalactiae in the PCR reaction system is 0.25 μmol / L; the final concentration of the upstream primer for the internal control gene and the downstream primer for the internal control gene in the PCR reaction system is 0.5 μmol / L; the final concentration of the probe for the internal control gene in the PCR reaction system is 0.25 μmol / L.

6. The kit for quantitatively detecting group B streptococcus nucleic acid according to claim 4, wherein The components of the GBS positive control product include a group B streptococcus pseudovirus with a concentration of not less than 1×10 5 copies / mL and a pseudovirus containing an internal standard fragment with a concentration of not less than 1×10 5 copies / mL; The components of the GBS negative control product include a pseudovirus containing an internal standard fragment with a concentration of not less than 5×10 4 copies / mL and TE buffer.

7. The kit for quantitatively detecting group B streptococcus nucleic acid according to claim 4, characterized in that The detection sample of the kit is human sputum; The criteria for the kit to determine a valid detection include: In each detection, only the reaction wells with a droplet count ≥ 10000 in all reaction wells are valid reaction wells; In the valid reaction wells, there should be a negative control group and a positive control group. When the test result of the positive control group is positive, the test result of the negative control group is negative, and the concentration test result of the positive control group is 10 5 ±10% copies / mL, the test result is valid.

8. A detection method for quantitatively detecting group B streptococcus nucleic acid for non-diagnostic purposes, which is detected using the kit according to any one of claims 4 to 7, characterized in that, Comprising the following steps: Collect a sample to be tested, extract nucleic acid from the sample to be tested to obtain nucleic acid of the sample to be tested; Take out the primer-probe mixture and the ddPCR premix from the kit, melt them at room temperature and mix well, prepare a PCR reaction system, and dispense the PCR reaction system into a preset number of PCR reaction tubes; Add the nucleic acid of the sample to be tested, the GBS positive control product and the GBS negative control product into the PCR reaction tubes respectively, perform a transient centrifugation and then transfer them to the droplet preparation area; Perform droplet preparation on the liquid in the PCR reaction tubes, and perform a PCR amplification reaction on the prepared droplets according to the preset PCR reaction conditions to obtain a PCR reaction product; Perform quantitative analysis on the PCR reaction product to obtain the copy number of Streptococcus agalactiae nucleic acid.

9. The detection method for quantitatively detecting group B streptococcus nucleic acid according to claim 8, wherein, The PCR reaction conditions are as follows: Enzyme heat activation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing at 58°C for 1 min, for a total of 45 cycles; finally, enzyme inactivation at 98°C for 10 min.

10. The detection method for quantitatively detecting group B streptococcus nucleic acid according to claim 8, characterized in that, The detection method further includes: Diluting Streptococcus agalactiae reference products S1-S3 with RNase-free deionized water at ratios of 1:10, 1:100, 1:1000, 1:10000, and 1:100000, respectively.

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