Primer probe combination, kit and detection method for quantitatively detecting haemophilus influenzae

Abstract

The invention belongs to the field of biological detection, and relates to a primer probe combination, a kit and a detection method for quantitatively detecting haemophilus influenzae, primers comprise an upstream primer for detecting haemophilus influenzae, a downstream primer for detecting haemophilus influenzae, an internal control upstream primer and an internal control downstream primer, the probe comprises a haemophilus influenzae detection probe and an internal control gene probe; the nucleotide sequence of the upstream primer for detecting haemophilus influenzae is as shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer for detecting haemophilus influenzae is as shown in SEQ ID NO: 2; the nucleotide sequence of the internal control upstream primer is as shown in SEQ ID NO: 3, and the nucleotide sequence of the internal control downstream primer is as shown in SEQ ID NO: 4; the nucleotide sequence of the haemophilus influenzae detection probe is as shown in SEQ ID NO: 5, and the nucleotide sequence of the internal control gene probe is as shown in SEQ ID NO: 6. According to the application, the qualitative and quantitative detection of haemophilus influenzae can be realized at the same time, and the sensitivity and accuracy of haemophilus influenzae detection are improved.

Description

Technical Field

[0001] The present application relates to the field of biological detection technologies, and in particular, to a primer-probe combination, a kit, and a detection method for quantitatively detecting Haemophilus influenzae. Background Art

[0002] Haemophilus influenzae (HI) is a Gram-negative bacillus without spores and flagella, showing various morphologies such as rod-shaped, coccobacillary, and filamentous, and colonizes in the nasopharynx of humans. The infection of Haemophilus influenzae occurs throughout the year but is relatively concentrated in winter and spring, often causing community-acquired respiratory infections, pneumonia, bronchitis, acute otitis media, bacterial meningitis and other diseases, and in severe cases, it can cause systemic infections such as suppurative meningitis and septicemia.

[0003] Research shows that infants and the elderly are the highest-risk groups, among which individuals with low immunity and underlying comorbidities are the most susceptible populations and are associated with high mortality rates. Therefore, how to control the infection of HI is an important public health issue.

[0004] Since the early clinical symptoms of Haemophilus influenzae are not typical and are similar to those of other respiratory pathogen infections, it is difficult to make an effective diagnosis through clinical symptoms, etc., and it is necessary to judge in combination with the results of clinical laboratory tests. At present, the common detection methods for respiratory pathogens include bacterial isolation and culture, antigen-antibody detection, sequencing / high-throughput sequencing, fluorescence PCR, etc., and these detection methods also play an important role in the detection of Haemophilus influenzae.

[0005] Bacterial isolation and culture is the gold standard for bacterial identification, but due to its long culture cycle, harsh culture conditions, and easy occurrence of false negative results, it has limitations in practical applications; antigen-antibody detection is divided into antigen detection and antibody detection. Antigen detection mainly uses direct immunofluorescence or serological diagnosis. Serological diagnosis has problems such as cumbersome operation, long time consumption, and easy occurrence of non-specific agglutination reactions. The direct immunofluorescence method also has problems such as difficult sampling and high cost. Antibody detection technologies such as colloidal gold and ELISA have low specificity and are more likely to produce false positive detection results; high-throughput sequencing is costly and not applicable to samples with low throughput; while fluorescence PCR can judge whether the target pathogen exists according to the change of the fluorescence characteristics of the reaction system, but this method is a qualitative or semi-quantitative detection and it is difficult to perform absolute quantitative detection of Haemophilus influenzae in the sample. Summary of the Invention

[0006] The purpose of the present application is to provide a primer-probe combination, a kit, and a detection method for quantitatively detecting Haemophilus influenzae, so as to solve the technical problem in the prior art that the Haemophilus influenzae in the sample cannot be quantitatively detected quickly and accurately.

[0007] To solve the above technical problems, the embodiments of the present application provide a primer-probe combination for quantitatively detecting Haemophilus influenzae, and adopt the following technical solutions:

[0008] The primers include an upstream primer for detecting Haemophilus influenzae, a downstream primer for detecting Haemophilus influenzae, an internal control upstream primer, and an internal control downstream primer, and the probes include a probe for detecting Haemophilus influenzae and an internal control gene probe;

[0009] The nucleotide sequence of the upstream primer for detecting Haemophilus influenzae is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer for detecting Haemophilus influenzae is shown in SEQ ID NO: 2; the nucleotide sequence of the internal control upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the internal control downstream primer is shown in SEQ ID NO: 4;

[0010] The nucleotide sequence of the probe for detecting Haemophilus influenzae is shown in SEQ ID NO: 5, and the nucleotide sequence of the internal control gene probe is shown in SEQ ID NO: 6.

[0011] Further, the 5' end of the nucleotide sequences corresponding to the probe for detecting Haemophilus influenzae and the internal control gene probe is labeled with a fluorescent group; the 3' end of the nucleotide sequences corresponding to the probe for detecting Haemophilus influenzae and the internal control gene probe is labeled with a quenching group;

[0012] Among them, the fluorescent group is selected from FAM, HEX or VIC; the quenching group is selected from MGB, BHQ1, BHQ2 or Eclipse.

[0013] Further, the 5' end of the nucleotide sequence of the probe for detecting Haemophilus influenzae is labeled with a FAM fluorescent group, and the 3' end of the nucleotide sequence of the probe for detecting Haemophilus influenzae is labeled with an MGB quenching group; the 5' end of the nucleotide sequence of the internal control gene probe is labeled with a VIC fluorescent group, and the 3' end of the nucleotide sequence of the internal control gene probe is labeled with an MGB quenching group.

[0014] To solve the above technical problems, the embodiments of the present application also provide a kit for quantitatively detecting Haemophilus influenzae, and adopt the following technical solutions:

[0015] It includes a primer-probe mixture, a premix, a HI positive control product, and a HI negative control product; wherein, the primer-probe mixture includes the primer-probe combination for quantitatively detecting Haemophilus influenzae as described above.

[0016] Further, the final concentrations of the upstream primer for detecting Haemophilus influenzae and the downstream primer for detecting Haemophilus influenzae in the PCR reaction system are 0.80 - 0.90 μmol / L respectively; the final concentration of the probe for detecting Haemophilus influenzae in the PCR reaction system is 0.20 - 0.30 μmol / L;

[0017] The final concentrations of the internal control upstream primer and the internal control downstream primer in the PCR reaction system are 0.80 - 0.90 μmol / L respectively; the final concentration of the internal control gene probe in the PCR reaction system is 0.20 - 0.30 μmol / L.

[0018] Further, the components of the HI positive control product include pseudovirus containing the Haemophilus influenzae gene fragment and pseudovirus containing the internal standard fragment; the components of the HI negative control product include pseudovirus containing the internal standard fragment and TE buffer.

[0019] Further, the primer - probe mixture further includes Tris - HCl buffer.

[0020] To solve the above - mentioned technical problems, the embodiment of the present application also provides a detection method for quantitatively detecting Haemophilus influenzae. Using the kit as described above for detection, the following technical solutions are adopted:

[0021] Collect a sample to be tested, extract nucleic acid from the sample to be tested to obtain the nucleic acid of the sample to be tested;

[0022] Take out the primer - probe mixture and the premixed solution 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;

[0023] Correspondingly add the nucleic acid of the sample to be tested, the HI positive control product and the HI negative control product into the PCR reaction tubes, centrifuge instantaneously and transfer them to the droplet preparation area;

[0024] Perform droplet preparation on the liquid in the PCR reaction tubes, and perform PCR amplification on the prepared droplets according to the PCR amplification conditions to obtain the PCR reaction product;

[0025] Perform quantitative analysis on the PCR reaction product to obtain the copy number of Haemophilus influenzae.

[0026] Further, the detection method further includes:

[0027] Take the sample to be tested with a concentration of (1.2 ± 10%)×10 7 copies / mL, and use RNase - free deionized water to perform 1:10, 1:10 2 、1:103 、1:10 4 Dilute to obtain test samples with different dilution concentrations;

[0028] Extract nucleic acids from the test samples with different dilution concentrations to obtain nucleic acids of the test samples with different dilution concentrations.

[0029] Furthermore, the reaction conditions for the PCR amplification are as follows:

[0030] After incubation at 50°C for 5 min; pre-denaturation 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.

[0031] Compared with the prior art, the present application mainly has the following beneficial effects:

[0032] The primer-probe combination, kit and detection method for quantitatively detecting Haemophilus influenzae provided by the present application are based on digital PCR technology to detect Haemophilus influenzae in a test sample, and can simultaneously achieve qualitative and quantitative detection of Haemophilus influenzae; in addition, the primer-probe combination of the present application has high specificity, reduces the misdiagnosis probability caused by non-specific binding, improves the sensitivity and accuracy of Haemophilus influenzae detection, and at the same time ensures the accuracy of the quantitative result of Haemophilus influenzae. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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 drawings in the following description 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.

[0034] Figure 1 is the fluorescence detection result of the negative control product detected by the kit for quantitatively detecting Haemophilus influenzae provided by the present application;

[0035] Figure 2 is the fluorescence detection result of the positive control product detected by the kit for quantitatively detecting Haemophilus influenzae provided by the present application;

[0036] Figures 3 to 6 are the ddPCR reaction results of the test samples S1 to S4 with different dilution concentrations in sequence;

[0037] Figures 7 to 16 are the detection results of the positive reference products P1 to P10;

[0038] Figures 17 to 26 are the detection results of the negative reference products N1 to N10. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one 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 "comprising" and "having" and any variations thereof in the specification and claims of this application and the above drawings are intended to cover non-exclusive inclusion. The terms "first", "second", etc. in the specification and claims of this application or the above drawings are used to distinguish different objects and not to describe a specific order.

[0040] Reference herein to "an embodiment" 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.

[0041] In order to enable those skilled in the technical field 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.

[0042] This application provides a primer-probe combination for quantitatively detecting Haemophilus influenzae. The primers in the primer-probe combination include an upstream primer for detecting Haemophilus influenzae (HI upstream primer), a downstream primer for detecting Haemophilus influenzae (HI downstream primer), an internal control upstream primer, and an internal control downstream primer. The probes in the primer-probe combination include a probe for detecting Haemophilus influenzae (HI probe) and an internal control gene probe.

[0043] As shown in Table 1, the nucleotide sequence of the HI upstream primer is as shown in SEQ ID NO: 1, and the nucleotide sequence of the HI downstream primer is as shown in SEQ ID NO: 2; the nucleotide sequence of the internal control upstream primer is as shown in SEQ ID NO: 3, and the nucleotide sequence of the internal control downstream primer is as shown in SEQ ID NO: 4. The nucleotide sequence of the HI probe is as shown in SEQ ID NO: 5, and the nucleotide sequence of the internal control gene probe is as shown in SEQ ID NO: 6.

[0044] Table 1 Nucleotide sequence table of the primer-probe combination for detecting Haemophilus influenzae

[0045]

[0046]

[0047] It should be noted that the nucleotide sequences corresponding to the HI upstream primer, HI downstream primer, and HI probe detect the detection site of the OmpP6 region of Haemophilus influenzae. The nucleotide sequences should ensure that all genotypes of Haemophilus influenzae can be detected, and also ensure that the detection HI biological primer-probe combination has excellent detection sensitivity at this detection site.

[0048] The primer-probe combination for quantitatively detecting Haemophilus influenzae in this application is designed following the following principles:

[0049] (1) The primer length is controlled between 18 and 25 bp;

[0050] (2) The primer Tm value is between 52 and 60 °C, and the GC value is 40 - 60%;

[0051] (3) The Tm value of the probe is about 5 - 10 °C higher than that of the primer (including the Tm value change brought by the modification group);

[0052] (4) As much as possible, avoid continuous matching of 4 or more bases in the primer-probe itself and between primer-probes to minimize the generation of primer-probe secondary structures or dimers;

[0053] (5) The 3' end of the primer must not have continuous matching of more than 4 bases with other primer-probes;

[0054] (6) The length of the target fragment is between 70 and 200 bp, and sequences with shorter lengths are preferably selected;

[0055] (7) The fluorescent group of the probe can be FAM, VIC, or other groups with similar emission light spectra.

[0056] Based on the digital PCR technology and following the above design principles, specific primer-probes are designed on the OmpP6 gene of Haemophilus influenzae for quantitative detection. OmpP6 encodes a transport protein on the cell membrane. In this application, the NCBI database is used to query the OmpP6 sequence of Haemophilus influenzae, and multiple groups of primer-probes are designed based on its conservativeness and screened by sample verification, and the optimal combination and reaction system are obtained through repeated experiments. Among them, the optimal primer-probe combination is shown in Table 1.

[0057] Both ends of the above probe are labeled with a fluorescent group and a quenching group. Among them, the 5' end of the nucleotide sequences corresponding to the Haemophilus influenzae detection probe and the internal control gene probe is labeled with a fluorescent group; the 3' end of the nucleotide sequences corresponding to the Haemophilus influenzae detection probe and the internal control gene probe is labeled with a quenching group;

[0058] Among them, the fluorescent group is selected from FAM, HEX, or VIC; the quenching group is selected from MGB, BHQ1, BHQ2, or Eclipse.

[0059] It should be understood that the fluorescent groups labeled at the 5'-ends of the HI probe and the internal control gene probe should be different, so as to determine the detection result according to the fluorescence intensities of different fluorescence channels.

[0060] Furthermore, the 5'-end of the nucleotide sequence of the HI probe is labeled with a FAM fluorescent group, and the 5'-end of the nucleotide sequence of the internal control gene probe is labeled with a VIC fluorescent group. The 3'-ends of the nucleotide sequences corresponding to the HI probe and the internal control gene probe are respectively labeled with an MGB quenching group.

[0061] The above-mentioned primers for detecting HI and the HI probe labeled with the FAM fluorescent group can be used for the quantitative detection of Haemophilus influenzae. According to the presence or absence of positive droplets in the FAM channel and the number of positive and negative droplets, it can be determined whether Haemophilus influenzae is contained in the sample and calculate the concentration of the target fragment of Haemophilus influenzae in the sample. Among them, the target fragment is the DNA of Haemophilus influenzae or a DNA fragment of Haemophilus influenzae.

[0062] The primer-probe combination for quantitatively detecting Haemophilus influenzae in this application has been specifically optimized, reducing the misdiagnosis probability caused by non-specific binding, and an internal control gene detection system has been designed for monitoring and correction.

[0063] This application provides a kit for quantitatively detecting Haemophilus influenzae. The kit includes a primer-probe mixture, a premix, an HI positive control product, and an HI negative control product; among them, the primer-probe mixture includes the primer-probe combination for quantitatively detecting Haemophilus influenzae as described above.

[0064] Among them, the primer-probe mixture further includes a Tris-HCl buffer solution.

[0065] The PCR reaction system is prepared using the primer-probe mixture and the premix. The final concentrations of the upstream primer for detecting Haemophilus influenzae and the downstream primer for detecting Haemophilus influenzae in the PCR reaction system are respectively 0.80 - 0.90 μmol / L; the final concentration of the probe for detecting Haemophilus influenzae in the PCR reaction system is 0.20 - 0.30 μmol / L; the final concentrations of the upstream primer for the internal control and the downstream primer for the internal control in the PCR reaction system are respectively 0.80 - 0.90 μmol / L; the final concentration of the internal control gene probe in the PCR reaction system is 0.20 - 0.30 μmol / L.

[0066] Preferably, the final concentration of the HI upstream primer in the PCR reaction system is 0.82 μmol / L, and the final concentration of the HI downstream primer in the PCR reaction system is 0.82 μmol / L; the final concentration of the HI probe in the PCR reaction system is 0.22 μmol / L; the final concentration of the internal control upstream primer in the PCR reaction system is 0.82 μmol / L, and the final concentration of the internal control downstream primer in the PCR reaction system is 0.82 μmol / L; the final concentration of the internal control gene probe in the PCR reaction system is 0.22 μmol / L.

[0067] The premix can be the ddPCR Supermix for Probes (No dUTP) suitable for DNA sample droplet digital PCR provided by Bio-rad, with the product number 1863023 or 1863024.

[0068] The kit of the present application further includes reference substances, which include HI positive control and HI negative control. Among them, the components of the HI positive control include pseudovirus containing Haemophilus influenzae gene fragment and pseudovirus containing internal standard fragment, and the components of the HI negative control include pseudovirus containing internal standard fragment and TE buffer.

[0069] The detection sample applicable to the kit of the present application is human sputum.

[0070] The criteria for the kit of the present application to determine the detection effectiveness are as follows:

[0071] In each detection, only the reaction wells with the number of droplets ≥ 10,000 in all reaction wells are valid reaction wells;

[0072] Among the valid reaction wells, the negative control and the positive control should be included. When the detection result of the positive control is positive, the detection result of the negative control is negative, and the detection result of the concentration of the positive control is 10 5 ±15% copies / mL, the detection result of this time is valid.

[0073] Based on the above-mentioned kit for quantitatively detecting Haemophilus influenzae, the present application further provides a detection method for quantitatively detecting Haemophilus influenzae using the kit as described above. The detection method includes the following steps:

[0074] Step S10, collect the sample to be tested, extract nucleic acid from the sample to be tested, and obtain the nucleic acid of the sample to be tested.

[0075] Among them, the sample to be tested is human sputum.

[0076] Sputum: After asking the subject to cough deeply, collect the expectorated sputum into a 50 mL screw-cap plastic tube containing 3 mL of sampling solution. If the sputum is not collected in the sampling solution, 2 - 3 mL of sampling solution or an equal volume of sputum digestion solution can be added before the test.

[0077] Extract the sampling solution using a qualified human sputum extraction kit. Specifically, a nucleic acid extraction or purification reagent (registration number: Yue Sui Xie Bei 20170583) produced by Daan Gene Co., Ltd. can be used to extract and purify nucleic acids from the collected samples.

[0078] Step S20: Take out the primer-probe mixture and premix from the kit, melt them at room temperature and mix well to prepare a PCR reaction system, and dispense the PCR reaction system into a preset number of PCR reaction tubes.

[0079] Take out the primer-probe mixture and 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.

[0080] Among them, the preset number N = the number of test samples + HI negative control + HI positive control

[0081] The single-person PCR reaction system is shown in Table 2.

[0082] Table 2 Single-person PCR reaction system

[0083] Component Volume Primer-probe mixture 5 μL Premix 10 μL

[0084] Step S30: Add the nucleic acid of the test sample, HI positive control, and HI negative control to the PCR reaction tubes respectively, and transfer them to the droplet preparation area after instantaneous centrifugation.

[0085] Taking the above single-person PCR reaction system as an example, add 5 μL of the nucleic acid of the test sample, HI positive control, and HI negative control to the PCR reaction tubes after adding the PCR reaction system respectively. Tighten the tube caps, and transfer them to the droplet preparation area after instantaneous centrifugation for 15 seconds.

[0086] Step S40: Prepare droplets from the liquid in the PCR reaction tubes, and perform PCR amplification on the prepared droplets according to the PCR amplification conditions to obtain PCR reaction products.

[0087] Prepare droplets according to the instructions 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.

[0088] Specifically, mix the PCR reaction system with added samples using a vortex oscillator. Take out a droplet preparation chip, transfer a column (8 reaction systems) of the reaction system 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 the reaction system until all the reaction systems to be tested are processed.

[0089] Seal the 96-well plate with an aluminum mold using a heat sealer.

[0090] Transfer the sealed 96-well plate to a qualitative PCR instrument and set the cycles according to Table 3 to perform the PCR reaction.

[0091] Table 3 PCR reaction conditions

[0092]

[0093] After PCR is completed, take out the 96-well plate containing the PCR reaction product and transfer it to a droplet reader. Among them, the length of the PCR reaction product is generally 70 - 200 bp.

[0094] Step S50, perform quantitative analysis on the PCR reaction product to obtain the copy number of Haemophilus influenzae.

[0095] Set the droplet reading parameters according to the instruction manual. Note the fluorescence channel selection: select the FAM channel to detect Haemophilus influenzae nucleic acid and select the VIC channel to detect the internal standard (internal control gene). After setting is completed, start the droplet reading program.

[0096] 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 and the concentration meets the expectations, this test is valid.

[0097] See Figure 1 and Figure 2 The ddPCR reaction results of the Haemophilus influenzae HI negative quality control product and HI positive quality control product detected by the kit of the present application shown. Among them, Ch1 corresponds to the FAM fluorescence channel and Ch2 corresponds to the VIC fluorescence channel. As can be seen from the figure, in Figure 1 , no signal is detected through the Ch1 channel, and the internal control gene fragment is detected through the Ch2 channel; Figure 2 , the Haemophilus influenzae gene fragment and the internal control gene fragment are detected through the Ch1 channel and the Ch2 channel respectively.

[0098] If the test is valid, view the fluorescence scatter plot of Ch1 (FAM channel). After drawing the threshold line, view the concentration of Ch1 in copies / μL. Finally, combine the sample dilution factor to calculate the concentration of the original sample.

[0099] It should be noted that the detection principle of this application is as follows:

[0100] The kit of this application uses the droplet digital PCR technology of Bio-rad. Specific primers and probes are designed in the conserved region of the Haemophilus influenzae genome. The detection probe of Haemophilus influenzae is labeled with the FAM fluorescent group, and the internal standard gene is labeled with the VIC fluorescent group. After preparing the PCR reaction system and loading the samples, it is dispersed into tens of thousands of droplets. Each droplet can perform the PCR reaction simultaneously. After the PCR reaction is completed, a droplet reader is used to detect the fluorescence level of each droplet to determine the positivity or negativity of the droplet. After counting the positivity and negativity of all droplets, it can be determined whether there is Haemophilus influenzae nucleic acid in the sample. Further, based on the statistical results of the positive and negative droplets and combined with the statistical formula, the copy number of Haemophilus influenzae nucleic acid in the sample can be calculated.

[0101] The following will further illustrate the content of this application in more detail with specific examples and elaborate on this application, but these examples are by no means intended to limit this application.

[0102] Example 1

[0103] This example provides the composition, packaging, and quantity (96 person-times / box) of a kit for quantitatively detecting Haemophilus influenzae, as shown in Table 4.

[0104] Table 4

[0105]

[0106]

[0107] Example 2

[0108] 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:

[0109] Take an appropriate amount of the HI primer-probe mixture and ddPCR Supermix for Probes (No dUTP). After preparing the PCR reaction system in the above manner, transfer it into a PCR reaction tube; use a Haemophilus influenzae sample with a concentration of (1.2 ± 10%) × 10 7 copies / mL, and use RNase-free deionized water for 1:10, 1:10 2 、1:10 3 、1:10 4Dilute to obtain test samples with different dilution concentrations, which are respectively used as S1 to S4. After nucleic acid extraction, take 5 μL of the extraction solution and add it to an eight-well tube of the prepared PCR reaction system to make the total volume 20 μL. Tighten the eight-well lid, use an oscillator to mix vigorously for 15 seconds, transfer it to the droplet preparation area after instantaneous centrifugation for 15 seconds.

[0110] Among them, the concentration of the Haemophilus influenzae sample is preferably 1.2×10 7 copies / mL.

[0111] After preparing the droplets according to the droplet preparation method in 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 amplification reaction on the prepared droplets according to the PCR reaction conditions in Table 3.

[0112] After the reaction ends, the reaction results are shown in Table 5 and Figures 3 to 6 , Figures 3 to 6 They are the ddPCR reaction results of S1 to S4 in sequence.

[0113] Table 5 Detection results of sample detection range and sensitivity

[0114]

[0115] It can be seen from the above table results that when the sample concentration > 1200 copies / mL (i.e., 6 copies / reaction), the kit of the present application can detect positive Haemophilus influenzae. When the sample concentration is between 12000 and 1200000 copies / mL (i.e., 60 - 6000 copies / reaction), the quantitative test result of the kit of the present application has a difference of < 15% from the true concentration, indicating that the lowest detection limit of the kit of the present application is 1200 copies / mL and the kit has high sensitivity.

[0116] Example 3

[0117] This example detects the accuracy of the kit of the present application.

[0118] The kit of the present application detects the national reference products P01 - P10 and N01 - N10 of the nucleic acid detection reagent for Haemophilus influenzae. Take 5 μL of the extraction solution and add it to an eight-well tube of the PCR reaction system prepared in step S20 to make the total volume 20 μL. Tighten the eight-well lid, use an oscillator to mix vigorously for 15 seconds, transfer it to the droplet preparation area after instantaneous centrifugation for 15 seconds.

[0119] After preparing the droplets according to the droplet preparation method in 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 amplification reaction on the prepared droplets according to the PCR reaction conditions in Table 3.

[0120] After the reaction ended, the reaction results are shown in Table 6 and Figures 7 to 26 , Figures 7 to 16 are the test results of positive reference products P1 - P10, Figures 17 to 26 are the test results of negative reference products N1 - N10.

[0121] Table 6 Accuracy test results

[0122]

[0123]

[0124] According to the test results shown in the above table, the coincidence rate of the negative reference products is 100%, the coincidence rate of the positive reference products is 100%, and the positive and negative coincidence rate of the accuracy test results of each reference product is 100%. This indicates a high detection accuracy and accurate quantitative results, showing that the accuracy test of the kit of this application meets the requirements. In addition, the test result of the positive reference product is positive for Haemophilus influenzae, and the test result of the negative reference product is negative, further indicating a high accuracy of the kit.

[0125] Example 4

[0126] This example is an application experiment, using the kit of this application and a third-party Haemophilus influenzae detection kit to detect the positive and negative of the test samples.

[0127] Twenty positive samples of Haemophilus influenzae and five negative samples were respectively selected. After nucleic acid extraction, the samples were labeled and the label information was ensured to be correct, and then stored at -80 °C. During the experiment, 5 μL of each sample was taken and added to a reaction tube containing the PCR reaction solution prepared in step one to make the total volume 20 μL. It was vigorously shaken and mixed evenly for 15 seconds with an oscillator, and then transferred to the droplet preparation area after instantaneous centrifugation for 15 seconds.

[0128] After the droplets were prepared according to the droplet preparation method in step S40 above, the 96-well plate was sealed with an aluminum mold on a heat sealer, and the sealed 96-well plate was transferred to a qualitative PCR. The prepared droplets were subjected to PCR amplification reaction according to the PCR reaction conditions in Table 3.

[0129] After the reaction ended, the reaction results are shown in Table 7 and Figure 9 and Figure 10 , Figure 9 are the test results of positive samples No. 1 - 20, Figure 10 are the test results of negative samples No. 21 - 25.

[0130] Table 7 Sample test results of the kit of this application and the third-party kit

[0131]

[0132]

[0133] The above test results show that among 25 samples, 20 samples are HI positive and 5 samples are negative. The consistency between the test results of positive and negative samples and those of the third-party reagent kit reaches 100%. By using the reagent kit of the present application, it is possible to identify whether the tested population carries Haemophilus influenzae and perform quantitative detection on it, which is worthy of popularization and application.

[0134] Obviously, the embodiments described above are only a part of the embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they 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, for those skilled in the art, they 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 directly or indirectly using the content of the specification and drawings of the present application in other related technical fields is similarly within the scope of patent protection of the present application.

Claims

1. A primer-probe combination for quantitatively detecting Haemophilus influenzae, characterized in that, The primers include an upstream primer for detecting Haemophilus influenzae, a downstream primer for detecting Haemophilus influenzae, an internal control upstream primer, and an internal control downstream primer, and the probes include a probe for detecting Haemophilus influenzae and an internal control gene probe; The nucleotide sequence of the upstream primer for detecting Haemophilus influenzae is shown as SEQ ID NO: 1, and the nucleotide sequence of the downstream primer for detecting Haemophilus influenzae is shown as SEQ ID NO: 2; the nucleotide sequence of the internal control upstream primer is shown as SEQ ID NO: 3, and the nucleotide sequence of the internal control downstream primer is shown as SEQ ID NO: 4; The nucleotide sequence of the probe for detecting Haemophilus influenzae is shown as SEQ ID NO: 5, and the nucleotide sequence of the internal control gene probe is shown as SEQ ID NO:

6.

2. The primer-probe combination for quantitatively detecting Haemophilus influenzae according to claim 1, characterized in that, The 5' ends of the nucleotide sequences corresponding to the probe for detecting Haemophilus influenzae and the internal control gene probe are labeled with a fluorescent group; the 3' ends of the nucleotide sequences corresponding to the probe for detecting Haemophilus influenzae and the internal control gene probe are labeled with a quenching group; Among them, the fluorescent group is selected from FAM, HEX or VIC; the quenching group is selected from MGB, BHQ1, BHQ2 or Eclipse.

3. The primer-probe combination for quantitatively detecting Haemophilus influenzae according to claim 2, characterized in that, The 5' end of the nucleotide sequence of the probe for detecting Haemophilus influenzae is labeled with a FAM fluorescent group, and the 3' end of the nucleotide sequence of the probe for detecting Haemophilus influenzae is labeled with an MGB quenching group; the 5' end of the nucleotide sequence of the internal control gene probe is labeled with a VIC fluorescent group, and the 3' end of the nucleotide sequence of the internal control gene probe is labeled with an MGB quenching group.

4. A kit for quantitatively detecting Haemophilus influenzae, characterized in that, It includes a primer-probe mixture, a premix, an HI positive control product, and an HI negative control product; among them, the primer-probe mixture includes the primer-probe combination for quantitatively detecting Haemophilus influenzae according to any one of claims 1 to 3.

5. The kit for quantitatively detecting Haemophilus influenzae according to claim 4, characterized in that, The final concentrations of the upstream primer for detecting Haemophilus influenzae and the downstream primer for detecting Haemophilus influenzae in the PCR reaction system are 0.80 - 0.90 μmol / L respectively; the final concentration of the probe for detecting Haemophilus influenzae in the PCR reaction system is 0.20 - 0.30 μmol / L; The final concentrations of the internal control upstream primer and the internal control downstream primer in the PCR reaction system are 0.80 - 0.90 μmol / L respectively; the final concentration of the internal control gene probe in the PCR reaction system is 0.20 - 0.30 μmol / L.

6. The kit for quantitatively detecting Haemophilus influenzae according to claim 4, characterized in that, The components of the HI positive control product include a pseudovirus containing a Haemophilus influenzae gene fragment and a pseudovirus containing an internal standard fragment; the components of the HI negative control product include a pseudovirus containing an internal standard fragment and a TE buffer solution.

7. The kit for quantitatively detecting Haemophilus influenzae according to claim 4, characterized in that, The primer-probe mixture further includes a Tris-HCl buffer solution.

8. A detection method for quantitatively detecting Haemophilus influenzae, using the kit according to any one of claims 4 to 7 for detection, characterized in that, It includes 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 premix from the kit, melt them at room temperature and mix them evenly to prepare a PCR reaction system, and divide the PCR reaction system into a preset number of PCR reaction tubes; Add the nucleic acid of the sample to be tested, the HI positive control product, and the HI negative control product to the PCR reaction tube respectively, and transfer them to the droplet preparation area after instantaneous centrifugation; Prepare droplets from the liquid in the PCR reaction tube, and perform PCR amplification on the prepared droplets according to the PCR amplification conditions to obtain a PCR reaction product; Perform quantitative analysis on the PCR reaction product to obtain the copy number of Haemophilus influenzae.

9. The detection method for quantitatively detecting Haemophilus influenzae according to claim 8, characterized in that, The detection method further includes: Take the test sample with a concentration of (1.2 ± 10%) × 10 7 copies / mL, and use RNase-free deionized water to perform 1:10, 1:10 2 , 1:10 3 , 1:10 4 dilution on the test sample to obtain test samples with different dilution concentrations; Extract nucleic acids from the samples to be tested at different dilution concentrations to obtain nucleic acids of the samples to be tested at different dilution concentrations.

10. The detection method for quantitatively detecting Haemophilus influenzae according to claim 8, characterized in that, The reaction conditions for the PCR amplification are as follows: After incubation at 50°C for 5 min; pre-denaturation 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.

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