Kit for detecting macrolide antibiotic-resistant bordetella pertussis and application thereof

Abstract

The invention relates to a primer probe group, a kit and a method for detecting macrolide antibiotic resistant bordetella pertussis, and particularly, primers and probes aim at the 2047 site of a 23S rRNA (ribosomal Ribonucleic Acid) gene of bordetella pertussis. The inventor optimizes the reaction conditions (annealing temperature, primer concentration and the like) of the PCR amplification reaction, so that the sensitivity and specificity of the kit and the method are improved, and the result accuracy is ensured.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a kit for detecting macrolide antibiotic-resistant Bordetella pertussis and application thereof. Background Art

[0002] Bordetella pertussis, also known as Bordetella pertussis, belongs to the Burkholderiales, Alcaligenesaceae, Bordetella genus. Pertussis is an acute respiratory infectious disease caused by Bordetella pertussis. People of all ages can be infected and develop the disease. It is one of the infectious diseases with high morbidity and mortality in infants and young children. Current studies have shown that Bordetella pertussis is highly sensitive to macrolide antibiotics, and is recommended as the first choice for the treatment of pertussis by various domestic and international guidelines. However, in recent years, Bordetella pertussis has developed resistance to macrolide antibiotics.

[0003] The mechanism of resistance of Bordetella pertussis to macrolide antibiotics is mainly due to the A2047G mutation in the central loop V domain of its 23S rRNA gene, which leads to a decrease in the affinity of macrolide antibiotics to the action site. It is relatively difficult to culture Bordetella pertussis. Among the existing technologies, traditional antibiotic sensitivity tests (such as the Kirby-Bauer method) and molecular biological methods (such as PCR amplification of resistance genes) are mainly used to diagnose whether a patient is infected with macrolide-resistant Bordetella.

[0004] There are many limitations in the detection of macrolide antibiotic-resistant Bordetella pertussis in the existing technology. 1. Differences in sensitivity: Different detection methods (such as E-test method, KB disc diffusion method, etc.) may have differences in sensitivity when determining the minimum inhibitory concentration (MIC) of the drug. This may lead to deviations in the judgment of the drug resistance of the same strain. For example, although the E-test method is more accurate, it is more expensive and less clinically carried out; while the KB disc diffusion method is easy to operate, but its sensitivity may not be as good as the E-test method. 2. Coexistence of multiple resistance mechanisms: The resistance mechanism of Bordetella pertussis to macrolide antibiotics may involve multiple factors, such as the acquisition of methylase genes. The coexistence of these mechanisms makes the diagnosis of drug resistance more complicated. 3. Transfer of resistance genes: Some resistance genes (such as ermA, ermB, etc.) can be horizontally transferred between different strains, which increases the risk of transmission of resistant strains and the difficulty of diagnosis. 3. Strain variability: The strains of Bordetella pertussis are highly variable, and there may be significant differences in drug resistance between different strains. This makes it difficult to generalize the drug resistance diagnosis results based on a single strain to all strains. 4. Regional differences: The distribution and drug resistance of Bordetella pertussis strains may vary between different regions. For example, there is a statistical difference in the resistance of clinically isolated Bordetella pertussis to macrolide antibiotics in Xi'an and Shanghai. This regional difference increases the complexity of drug resistance diagnosis.

[0005] Therefore, there is an urgent need in the art for a rapid, accurate and simple method for detecting the A2047G mutation of Bordetella pertussis resistance to macrolide antibiotics. Summary of the invention

[0006] The purpose of the present invention is to provide a method for rapidly, accurately and conveniently detecting the A2047G mutation of Bordetella pertussis resistance to macrolide antibiotics.

[0007] In a first aspect of the present invention, a primer probe set for detecting Bordetella pertussis is provided, the primer probe set comprising:

[0008] (i) an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; and

[0009] (ii) a probe selected from the group consisting of:

[0010] (a) a first probe having a nucleotide sequence as shown in SEQ ID NO: 3 and a probe having a nucleotide sequence as shown in SEQ ID NO:

[0011] 4. A second probe having a nucleotide sequence as shown in FIG.

[0012] (b) a third probe having a nucleotide sequence as shown in SEQ ID NO: 5 and a third probe having a nucleotide sequence as shown in SEQ ID NO:

[0013] A fourth probe having the nucleotide sequence shown in 6;

[0014] (c) a fifth probe having a nucleotide sequence as shown in SEQ ID NO: 7 and a fifth probe having a nucleotide sequence as shown in SEQ ID NO:

[0015] The sixth probe has the nucleotide sequence shown in 8.

[0016] In another preferred embodiment, the probe is modified with a fluorescent group and a quencher group.

[0017] In another preferred embodiment, the first probe and the second probe are modified with different fluorescent groups, the third probe and the fourth probe are modified with different fluorescent groups, and the fifth probe and the sixth probe are modified with different fluorescent groups.

[0018] In another preferred embodiment, the fluorescent group is selected from the following group: FAM, HEX, VIC, ROX, CY5, or a combination thereof.

[0019] In another preferred embodiment, the quenching group is selected from the following group: BHQ1, BHQ2, BHQ3, MGB, TAMRA, DABCYL, or a combination thereof.

[0020] In another preferred embodiment, the primer probe set includes:

[0021] (i) an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; and

[0022] (ii) a first probe having a nucleotide sequence as shown in SEQ ID NO:3 and a second probe having a nucleotide sequence as shown in SEQ ID NO:4.

[0023] In another preferred embodiment, the Bordetella pertussis is Bordetella pertussis resistant to macrolide antibiotics.

[0024] In a second aspect, the present invention provides a kit, wherein the kit comprises the primer probe set as described in the first aspect of the present invention.

[0025] In another preferred embodiment, the first probe and the second probe in the primer probe set are not in the same container; the third probe and the fourth probe in the primer probe set are not in the same container; the fifth probe and the sixth probe in the primer probe set are not in the same container.

[0026] In another preferred embodiment, the kit further comprises a PCR premix, wherein the PCR premix comprises a DNA polymerase, dNTPs and Mg 2+ .

[0027] In another preferred example, the PCR premix further includes a PCR buffer.

[0028] In another preferred embodiment, the kit further comprises a positive quality control product and a negative quality control product.

[0029] In another preferred embodiment, the positive quality control product includes the DNA of a mutant strain of Bordetella pertussis.

[0030] In another preferred embodiment, the negative control product is selected from the following group: nucleic acid of Escherichia coli, Streptococcus pneumoniae, Haemophilus influenzae, or a combination thereof.

[0031] In another preferred embodiment, the kit is used to detect Bordetella pertussis.

[0032] In a third aspect, the present invention provides a method for detecting Bordetella pertussis, the method comprising the steps of:

[0033] (s1) providing a sample to be tested, the primer probe set as described in the first aspect of the present invention, and a PCR premix solution, wherein the PCR premix solution includes a buffer, a DNA polymerase, dNTPs, and Mg 2+ ;

[0034] (s2) Mixing the sample to be tested, the primer probe set and the PCR premix to obtain a PCR reaction solution, and performing a PCR reaction to obtain a test result.

[0035] In another preferred embodiment, the molar ratio of the upstream primer to the downstream primer is 1:10 to 1:200, preferably 1:30 to 1:180, and more preferably 1:50 to 1:100.

[0036] In another preferred embodiment, the concentration of the upstream primer and the downstream primer in the PCR reaction solution is independently 0.01 to 500 μM, preferably 0.05 to 100 μM, and more preferably 0.1 to 50 μM.

[0037] In another preferred embodiment, the concentration of the probe in the PCR reaction solution is 0.01-5 μM, preferably 0.05-1 μM, and more preferably 0.1-0.4 μM.

[0038] In another preferred embodiment, the reaction procedure of the PCR reaction is 50°C oil phase solidification for 3 to 10 min; 95°C pre-denaturation for 3 to 10 min; 95°C denaturation for 5 to 30 s, 58°C annealing and extension for 30 to 60 s, and 40 to 50 cycles.

[0039] In another preferred embodiment, the reaction procedure of the PCR reaction is 50°C oil phase solidification for 4 to 8 min; 95°C pre-denaturation for 4 to 8 min; 95°C denaturation for 5 to 20 s, 58°C annealing and extension for 30 to 50 s, 43 to 48 cycles;

[0040] Preferably, the oil phase is solidified at 50°C for 5 min; pre-denaturation is performed at 95°C for 5 min; denaturation is performed at 95°C for 10 s, and annealing and extension is performed at 58°C for 40 s, for 45 cycles.

[0041] In another preferred embodiment, the volume of the sample to be tested is 1-20 μl, preferably 5-20 μl, more preferably 10-20 μl, for example, about 15 μl.

[0042] In another preferred embodiment, the concentration of the DNA polymerase in the PCR reaction solution is 0.1-10U, preferably 0.5-8U, more preferably 0.8-5U, for example, about 3.5U.

[0043] In another preferred example, the primer probe set is premixed with the PCR premix solution.

[0044] In another preferred embodiment, the buffer is 3×PCR buffer.

[0045] In another preferred embodiment, the Bordetella pertussis is Bordetella pertussis resistant to macrolide antibiotics.

[0046] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0047] In another preferred embodiment, the sensitivity of the method is ≤100 copies / ml, preferably ≤50 copies / ml.

[0048] In another preferred example, the PCR reaction is digital PCR.

[0049] In another preferred embodiment, the first detection result corresponds to the signal value of the fluorescent group modified by the first, third or fifth probe, and the second detection result corresponds to the signal value of the fluorescent group modified by the second, fourth or sixth probe.

[0050] In another preferred embodiment, the judgment criteria of the method are:

[0051] When the number of positive points in the first test result is ≥10 and the number of positive points in the second test result is <3, the sample to be tested is a mutant strain sample of Bordetella pertussis;

[0052] When the number of positive points in the first test result is less than 3 and the number of positive points in the second test result is greater than 10, the sample to be tested is a wild strain sample of Bordetella pertussis.

[0053] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Detection results of the optimized probe are shown.

[0055] Figure 2 The specific results of the method of the present invention are shown.

[0056] Figure 3 The feasibility verification of the method of the present invention is shown, A: mutant sample; B: wild strain sample.

[0057] Figure 4 The results of optimizing primer concentrations are shown: A: the concentration ratio of upstream and downstream primers is 50:1; B: the concentration of upstream primer: downstream primer is 1:50.

[0058] Figure 5 The results of optimizing the annealing temperature in the PCR program are shown, A: the detection result of the wild strain sample with an annealing temperature of 55°C; B: the detection result of the mutant strain sample with an annealing temperature of 58°C; C: the detection result of the wild strain sample with an annealing temperature of 58°C; D: the detection result of the wild strain sample with an annealing temperature of 62°C.

[0059] Figure 6Sequencing results of clinical samples are shown.

[0060] Figure 7 The results of digital PCR testing of clinical samples are shown.

[0061] Figure 8 The results of digital PCR sample sensitivity detection are shown. DETAILED DESCRIPTION

[0062] The inventors, through extensive and in-depth research, a large number of experiments and screening, unexpectedly discovered for the first time a specific primer and probe at position 2047 of the 23SrRNA gene. Thus, a primer probe set, a kit and a method for detecting macrolide antibiotic-resistant Bordetella pertussis were developed. The inventors improved the sensitivity and specificity of the kit and the method by optimizing the reaction conditions annealing (temperature and primer concentration, etc.) of the probe sequence and the PCR amplification reaction, thereby ensuring the accuracy of the results. The present invention was completed on this basis.

[0063] the term

[0064] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0065] As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., are understood to include the stated elements or components but do not exclude other elements or components.

[0066] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0067] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range and fractional values ​​thereof (e.g., one tenth and one hundredth of an integer) where appropriate.

[0068] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0069] The term "probe" has the meaning commonly understood by those skilled in the art, that is, a short single-stranded DNA or RNA fragment used to detect a nucleic acid sequence complementary thereto.

[0070] As used herein, the terms "FAM signal" and "first detection result" are used interchangeably, and both refer to the detection result of detecting the sample to be tested using the upstream primer, downstream primer and the probe shown in SEQ ID NO: 3, 5 or 7 in the primer probe set of the present invention.

[0071] As used herein, the terms "HEX signal" and "second detection result" are used interchangeably, and both refer to the detection result of detecting the sample to be tested using the upstream primer, downstream primer and the probe shown in SEQ ID NO: 4, 6 or 8 in the primer probe set of the present invention.

[0072] Macrolide-resistant Bordetella pertussis

[0073] Bordetella pertussis is the pathogen of whooping cough, which is transmitted through respiratory droplets or close contact. Infection can cause symptoms such as paroxysmal spasmodic cough, and the course of the disease may last for weeks or even months.

[0074] Macrolide antibiotics are a class of drugs with antibacterial activity, usually used to treat infections caused by sensitive bacteria. In the treatment of pertussis, macrolide antibiotics such as azithromycin, erythromycin, roxithromycin or clarithromycin were once widely used as the first choice of drugs.

[0075] "Macrolide-resistant Bordetella pertussis" refers to a type of Bordetella pertussis that is resistant to macrolide antibiotics. After these bacteria infect the human body, their resistance to macrolide antibiotics may lead to treatment failure or prolonged course of the disease.

[0076] Primer probe set of the present invention

[0077] The primer probe set of the present invention comprises

[0078] (i) an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; and

[0079] (ii) a probe selected from the group consisting of:

[0080] (a) a first probe having a nucleotide sequence as shown in SEQ ID NO: 3 and a probe having a nucleotide sequence as shown in SEQ ID NO:

[0081] 4. A second probe having a nucleotide sequence as shown in FIG.

[0082] (b) a third probe having a nucleotide sequence as shown in SEQ ID NO: 5 and a third probe having a nucleotide sequence as shown in SEQ ID NO:

[0083] A fourth probe having the nucleotide sequence shown in 6;

[0084] (c) a fifth probe having a nucleotide sequence as shown in SEQ ID NO: 7 and a fifth probe having a nucleotide sequence as shown in SEQ ID NO:

[0085] The sixth probe has the nucleotide sequence shown in 8.

[0086] In a preferred embodiment, the probe is modified with a fluorescent group and a quencher group.

[0087] In a preferred embodiment, the primer probe set comprises:

[0088] (i) an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; and

[0089] (ii) a first probe having a nucleotide sequence as shown in SEQ ID NO:3 and a second probe having a nucleotide sequence as shown in SEQ ID NO:4.

[0090] The primer probe set of the present invention uses the conservative region of 23S rRNA of Bordetella pertussis as the detection target gene, targets the 2047 site of the 23S rRNA gene, does not produce nonspecific amplification in nucleic acid amplification, has excellent specificity, and makes the detection result accurate.

[0091] Method of the present invention

[0092] The method of the present invention is used to detect the drug resistance of Bordetella pertussis to macrolide antibiotics, comprising the steps of:

[0093] (s1) providing a sample to be tested, the primer probe set as described in the first aspect of the present invention, and a PCR premix solution, wherein the PCR premix solution includes a DNA polymerase, dNTPs, and Mg 2+ ;

[0094] (s2) Mixing the sample to be tested, the primer probe set and the PCR premix to obtain a PCR reaction solution, and performing a PCR reaction to obtain a test result.

[0095] In a preferred embodiment, the molar ratio of the upstream primer to the downstream primer is 1:10 to 1:200, preferably 1:30 to 1:180, and more preferably 1:50 to 1:100.

[0096] In a preferred embodiment, the concentration of the probe in the PCR reaction solution is 0.01 to 5 μM, preferably 0.05 to 1 μM, and more preferably 0.1 to 0.4 μM.

[0097] In a preferred embodiment, the reaction procedure of the PCR reaction is 50°C oil phase solidification for 3-10 min; 95°C pre-denaturation for 3-10 min; 95°C denaturation for 5-30 s, 58°C annealing and extension for 30-60 s, and 40-50 cycles.

[0098] In a preferred embodiment, the concentration of the DNA polymerase in the PCR reaction solution is 0.1-10U, preferably 0.5-8U, more preferably 0.8-5U, for example, about 3.5U.

[0099] In a preferred embodiment, the primer probe set is premixed with the PCR premix solution.

[0100] In a preferred embodiment, the judgment criteria of the method are: if the number of FAM positive points in the mutation signal channel of the tested sample is greater than or equal to 10, and the number of HEX channel positive points is less than 3, then the sample is judged to be a mutant strain sample of Bordetella pertussis; if the number of HEX positive points in the wild signal channel is greater than 10, and the number of FAM channel positive points is less than 3, then the sample is judged to be a wild strain sample of Bordetella pertussis.

[0101] The method of the present invention uses an asymmetric primer probe to improve specificity and reduce the crossover problem between wild-type samples and mutant samples. The method of the present invention has excellent sensitivity and specificity at an annealing temperature of 57 to 59°C. In actual sample detection, the method of the present invention can detect Bordetella pertussis as low as 50 copies / ml, with excellent sensitivity.

[0102] The main advantages of the present invention include:

[0103] The kit or method of the present invention can quickly and accurately detect the drug resistance of Bordetella pertussis to macrolide antibiotics, and has the characteristics of good specificity and high sensitivity. The method of the present invention greatly shortens the detection time, and the sensitivity is higher than that of the conventional PCR detection method, and the detection sensitivity can reach 50 copies / ml, meeting the needs of clinical rapid diagnosis. The method of the present invention can also perform quantitative detection, and the degree of infection of the patient can also be reflected according to the accurate quantitative results, which is helpful for long-term tracking of the disease condition and better providing postoperative tracking and medication guidance for the patient.

[0104] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0105] Example 1 Design and screening of primer probes

[0106] 1.1 Methods

[0107] According to the pertussis Bordetella in the GenBank database, 23S rRNA conservative region is selected as the detection target gene, and the pertussis Bordetella bacterium and its drug-resistant mutation site are detected simultaneously. According to the primer design principle, primers and probes are designed, and the two are at least designed in three pairs, and different modification modes are used to compare feasibility. All primers and probes are retrieved by NCBI to ensure that non-specific amplification will not be carried out. The primers and probes used in the present embodiment are as shown in Table 1.

[0108] Table 1

[0109]

[0110] Note: Bold italics indicate mutation sites.

[0111] The PCR reaction system is a total system of 30 μl, including 10 μl of buffer (3×PCR Buffer), 1 μl of Taq enzyme (3.5U), 4 μl of primer-probe combination and 15 μl of DNA template, wherein the concentration of the upstream primer and the downstream primer is 0.4 μM, and the concentration of the probe is 0.2 μM.

[0112] The reaction procedure of ddPCR was as follows: 50°C oil phase solidification for 5 min; 95°C pre-denaturation for 5 min; 95°C denaturation for 10 sec, 58°C annealing and extension for 40 sec, and 45 cycles.

[0113] The DNA samples of the mutant strain of Bordetella pertussis were used as templates, and qPCR detection was performed with three sets of primers and probes to screen out the best primer and probe set.

[0114] Escherichia coli, Streptococcus pneumoniae, Haemophilus influenzae and other nucleic acid samples (5ul, concentration 10 5 copies / ml), and qPCR detection was performed with primers and the first set of probes to verify specificity.

[0115] Digital PCR was performed using primers and the first set of probes. The positive sample was the DNA of the mutant strain of Bordetella pertussis (the DNA concentration in the sample was 10 6 copies / ml), and the negative sample was a wild strain of Bordetella pertussis.

[0116] 1.2 Results

[0117] like Figure 1 As shown, only the first set of probes was able to distinguish the mutant strain DNA, so the first set of probes was selected for subsequent experiments.

[0118] like Figure 2 As shown, the results of using primers and the first set of probes to detect Escherichia coli, Streptococcus pneumoniae, and Haemophilus influenzae were all negative, and there was no cross-talk with wild-type Bordetella pertussis, indicating that the primer-probe set had good specificity.

[0119] like Figure 3 As shown in the figure, the difference between the negative and positive values ​​of the primers and the first set of probes during amplification is obvious, and the result is presented as a two-dimensional scatter plot, the upper shaded part is the gray value of the positive point, and the bottom shaded part is the gray value of the negative point; the gray value of the positive point is high, and the gray value of the negative point is low, and the Gap value between the negative point and the positive point represents the brightness difference between the positive and negative points. Therefore, the larger the Gap value between the positive and negative points, the higher the binding efficiency of the probe and the target gene, and the better the detection effect.

[0120] Example 2 Optimization of primer probe concentration

[0121] 2.1 Methods

[0122] Since it is difficult to distinguish between wild strains and mutant strains during the detection process, how to avoid cross-detection of primers and probes when detecting samples and to be able to specifically distinguish between wild strains and mutant strains is a major difficulty in this design. For this reason, a lot of optimization was carried out during the experiment.

[0123] The primer probe concentration is optimized by using a primer concentration asymmetric PCR method. A large amount of single-stranded DNA is obtained by guiding the amplification of a pair of oligonucleotide primers in unequal amounts. The amplification of specific and non-specific products can be effectively controlled by controlling the annealing temperature during the reaction.

[0124] In this embodiment, the molar ratio used is 1:50 to 1:100, and the key is the absolute amount of the limiting primer. The upstream primer: downstream primer molar ratio is 1:50 and the upstream: downstream primer ratio is 50:1. The wild strain DNA sample and the mutant strain DNA sample are used for preliminary verification on the digital PCR platform. The primers and the first set of probes described in Example 1 are used, and the specific steps are as shown in Example 1.

[0125] 2.2 Results

[0126] like Figure 4 As shown, the use of asymmetric primer probes at a molar ratio of 1:50 can significantly improve the specificity of the primer probes and reduce the crossover problem between wild-type samples and mutant samples.

[0127] Example 3 Optimization of annealing temperature

[0128] 3.1 Methods

[0129] The digital PCR platform can give quantitative results without the need for a standard curve, and this feature has certain advantages in the quantitative detection of nucleic acids.

[0130] The wild strain and mutant strain bacterial liquid (50 copies / mL) were respectively taken to extract DNA and used as templates to test the sensitivity of the primer probe digital PCR platform, and the reaction program was optimized to improve the detection rate of low-concentration samples. The annealing temperature was set to 55°C, 58°C, and 62°C, respectively, to test the detection of the kit in low-concentration samples. The primers and the first set of probes described in Example 1 were used, and the specific steps were as shown in Example 1.

[0131] 3.2 Results

[0132] like Figure 5 As shown in the figure, by using different annealing temperatures, the sensitivity and specificity were better when the annealing temperature was 58°C.

[0133] Example 4 Quantitative detection of clinically isolated strains using the method of the present invention

[0134] 4.1 Methods

[0135] This example uses Bordetella pertussis isolated and cultured from clinical samples, uses the Berger-00896B extraction kit to extract nucleic acid from 2 ml of sample bacterial solution, and performs quantitative detection by ddPCR. The total volume of the ddRT-PCR reaction system is 30 μl, and the specific preparation is as follows: 10 μl buffer (3×PCR Buffer), 1 μl Taq enzyme, 4 μl primer probe combination and 15 μl DNA template; the molar ratio of upstream primer: downstream primer is 1:50, and clinical samples are verified.

[0136] 4.2 Results

[0137] Table 2 Comparison of the method of the present invention and sequencing results

[0138] sample Sequencing results Digital PCR quantification results and concentration B1a Bordetella pertussis wild strain <![CDATA[Mutant strain (4.04×10 7 copies / ml)]]> 1D Bordetella pertussis mutant <![CDATA[Wild strain (9.7×10 6 copies / ml)]]>

[0139] Figure 6 The sequencing results of clinical sample test results are Figure 7The test results of digital PCR, where samples B1a and 1D are clinical samples of Bordetella pertussis, and B1e and 2D are negative controls.

[0140] As shown in Table 2, Figure 6 and Figure 7 As shown, the detection results of the method of the present invention are consistent with the sequencing results, indicating that the established ddRT-PCR method has excellent applicability and accuracy for detecting clinical samples and strains of Bordetella pertussis.

[0141] Example 5 Sensitivity of the method of the present invention

[0142] 5.1 Methods

[0143] This example uses Bordetella pertussis isolated and cultured from clinical samples (1A, 1B, 1C, 1D and 2A are mutant strains with 5 replicates, 2B, 2C, 2D, 3A and 3B are wild strains with 5 replicates, and 3C and 3D are negative controls). The bacterial solution is diluted to a detection limit of 50 copies / ml, and 2 ml of the diluted bacterial solution is extracted using a Bergey-00896B extraction kit for nucleic acid extraction, and quantitative detection is performed by ddPCR. The ddRT-PCR reaction system is specifically prepared as shown in Example 4.

[0144] 5.2 Results

[0145] like Figure 8 As shown, the kit and the procedure were used to detect low-concentration samples, and multiple repeated tests were able to detect the bacteria without crossover. The results showed that the method of the present invention can detect Bordetella pertussis at a concentration as low as 50 copies / ml, and has excellent reproducibility.

[0146] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A primer probe set for detecting Bordetella pertussis, characterized in that: The primer probe set comprises: (i) an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; and (ii) a probe selected from the group consisting of: (a) a first probe having a nucleotide sequence as shown in SEQ ID NO: 3 and a probe having a nucleotide sequence as shown in SEQ ID NO:

4. A second probe having a nucleotide sequence as shown in FIG. (b) a third probe having a nucleotide sequence as shown in SEQ ID NO: 5 and a third probe having a nucleotide sequence as shown in SEQ ID NO: A fourth probe having the nucleotide sequence shown in 6; (c) a fifth probe having a nucleotide sequence as shown in SEQ ID NO: 7 and a fifth probe having a nucleotide sequence as shown in SEQ ID NO: The sixth probe has the nucleotide sequence shown in 8.

2. The primer probe set according to claim 1, characterized in that: The probe is modified with a fluorescent group and a quenching group.

3. The primer probe set according to claim 1, characterized in that: The primer probe set comprises: (i) an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; and (ii) a first probe having a nucleotide sequence as shown in SEQ ID NO:3 and a second probe having a nucleotide sequence as shown in SEQ ID NO:

4.

4. A kit, characterized in that: The kit comprises the primer-probe set as claimed in claim 1.

5. The kit according to claim 4, characterized in that The kit also includes a PCR premix, which includes DNA polymerase, dNTPs and Mg 2+ .

6. A method for detecting Bordetella pertussis, characterized in that: The method comprises the steps of: (s1) providing a sample to be tested, the primer probe set as claimed in claim 1, and a PCR premix solution, wherein the PCR premix solution comprises a buffer, a DNA polymerase, dNTPs, and Mg 2+ ; (s2) Mixing the sample to be tested, the primer probe set and the PCR premix to obtain a PCR reaction solution, and performing a PCR reaction to obtain a test result.

7. The method according to claim 6, characterized in that The molar ratio of the upstream primer to the downstream primer is 1:10 to 1:

200.

8. The method according to claim 6, characterized in that The molar ratio of the upstream primer to the downstream primer is 1:30 to 1:

180.

9. The method according to claim 6, characterized in that The concentration of the probe in the PCR reaction solution is 0.01-5 μM, preferably 0.05-1 μM, and more preferably 0.1-0.4 μM.

10. The method according to claim 6, characterized in that The reaction procedure of the PCR reaction is 50° C. oil phase solidification for 3 to 10 minutes; 95° C. pre-denaturation for 3 to 10 minutes; 95° C. denaturation for 5 to 30 seconds, 58° C. annealing and extension for 30 to 60 seconds, and 40 to 50 cycles.

Images