Kit for detecting pertussis nucleic acid and drug-resistant gene through high-resolution melting curve and application method thereof

Abstract

The invention discloses a kit for detecting nucleic acid and drug-resistant genes of pertussis through a high-resolution melting curve and an application method of the kit, and the kit for detecting nucleic acid and drug-resistant gene loci of respiratory tract infection pertussis is developed by combining a PCR (Polymerase Chain Reaction) technology with a high-resolution melting curve analysis technology. The pertussis infection can be diagnosed in the early stage by only using one group of primer probes, and meanwhile, the drug resistance condition of the bordetella pertussis can be accurately judged. And a reliable experimental means is provided for clinical prevention and control of pertussis infection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a kit for detecting pertussis nucleic acid and drug-resistant genes by high-resolution melting curve and an application method thereof. Background Art

[0002] Bordetella pertussis is a member of the genus Bordetella in the family Alcaligenes. This bacterium is characterized by its rod shape, Gram-negative staining, obligate aerobicity, and non-fermentation. Pertussis produces a wide range of virulence factors, including a large number of toxins and bioactive products, which play a vital role in its pathogenesis and immune processes. The most important virulence factor is pertussis toxin (PT). It not only exhibits mitogenic activity and affects lymphocyte circulation, but also acts as an adhesin for the binding of bacteria to ciliated cells in the respiratory tract. The incubation period of pertussis is 2-21 days, generally 7-10 days. Typical cases include 3 clinical stages: catarrhal stage, spasmodic cough stage, and recovery stage. The course of the disease is about 6-12 weeks, and some cases may be longer. Newborns and infants under 6 months old often cause paroxysmal cyanosis after coughing, and in severe cases, apnea, convulsions, bradycardia or cardiac arrest. Adolescents, adults and some children may not have typical spasmodic coughs, but may present with chronic coughs that are not cured. Macrolide antibiotics such as erythromycin and azithromycin were once the first choice for the treatment of pertussis. However, in recent years, the resistance rate of clinical pertussis strains in China to macrolide antibiotics has reached more than 50%, so monitoring of resistance is extremely important. For children with drug resistance, consider treatment with trimethoprim-sulfamethoxazole or β-lactam antibiotics. Therefore, early diagnosis of Bordetella pertussis and drug resistance is of great significance for the prevention and treatment of pertussis infection in clinical practice.

[0003] Clinically, bacterial culture is the gold standard for diagnosing pertussis infection, but the culture process is time-consuming and has a high false-negative rate, making it impossible to achieve early diagnosis of pertussis infection. Fluorescence quantitative PCR detection is the most widely used technical method in clinical practice today. The advantages of this method are mainly high sensitivity and high specificity. Clinically, pertussis nucleic acid detection has been widely used in the diagnosis of pertussis, but there is no clinical kit for monitoring drug-resistant genes in clinical practice. Summary of the invention

[0004] The first object of the present invention is to provide a kit for detecting pertussis nucleic acid and drug-resistant gene using high-resolution melting curve in response to the above-mentioned problems existing in the prior art; the second object of the present invention is to provide a method for detecting pertussis nucleic acid and drug-resistant gene using the above-mentioned kit.

[0005] The first object of the present invention is achieved through the following technical scheme: a kit for detecting pertussis nucleic acid and drug-resistant gene using high-resolution melting curve, comprising a fluorescent PCR reaction solution, an enzyme solution, a wild-type Bordetella pertussis positive control substance, a drug-resistant Bordetella pertussis positive control substance, and a negative control substance, wherein the fluorescent PCR reaction solution contains a PCR buffer, Mgcl2, dNTPs, an upstream primer corresponding to the detection target, a downstream primer corresponding to the detection target, and a fluorescent probe corresponding to the detection target.

[0006] The detection target is the 23S rRNA gene;

[0007] The detection site is site 2047 of the 23S rRNA gene;

[0008] The sequence of the upstream primer is shown in SEQ ID NO: 1;

[0009] The sequence of the downstream primer is shown in SEQ ID NO: 2;

[0010] The sequence of the fluorescent probe is shown in SEQ ID NO:3.

[0011] Preferably, it also includes a box body, which is provided with a plurality of container holes, and the plurality of container holes are used to place fluorescent PCR reaction solution tubes, enzyme solution tubes, wild-type positive control substances, drug-resistant positive control substances, and negative control substance tubes respectively.

[0012] Preferably, the wild-type positive control and the drug-resistant positive control are mixed solutions of the detection target amplification sequence obtained by dissolving in TE buffer, and the amplification sequence of the detection target in the wild-type positive control is shown in SEQ ID NO: 4; the amplification sequence of the detection target in the drug-resistant positive control is shown in SEQ ID NO: 5.

[0013] Preferably, the negative control substance is sterile water for injection.

[0014] Preferably, the enzyme solution contains Taq DNA polymerase.

[0015] The second object of the present invention can be achieved by the following technical scheme: A method for detecting pertussis nucleic acid and drug resistance gene using high-resolution melting curve, comprising the following steps:

[0016] Step S01, nucleic acid extraction: extracting nucleic acid from the sample to be tested by magnetic bead method or column extraction method; Step S02, reagent preparation: taking PCR reaction solution and enzyme solution, preparing PCR qualitative reaction liquid;

[0017] Step S03, amplification detection: take the extraction product obtained in step S01, the wild-type positive control substance, the drug-resistant positive control substance, and the negative control substance, respectively add the PCR qualitative reaction liquid obtained in the above step S02, prepare the PCR system, and perform detection on a single-color fluorescence quantitative PCR instrument;

[0018] Step S04, performing fluorescence quantitative result analysis.

[0019] Preferably, in step S04, the fluorescence quantitative results are analyzed as follows:

[0020] When the peak temperature of the melting curve of the FAM fluorescence channel is 55.2±0.5℃, the 2047 position of the 23SrRNA gene of B. pertussis has not mutated, and it is judged as positive for B. pertussis nucleic acid;

[0021] When the peak temperature of the melting curve of the FAM fluorescence channel is 44.7±0.5℃, the 2047 position of the 23SrRNA gene of Bordetella pertussis is mutated; it is judged as positive for antibiotic-resistant Bordetella pertussis nucleic acid;

[0022] When no melting curve peak appears in the FAM fluorescence channel, it is judged as pertussis negative.

[0023] Preferably, in the step S02, 15 μL of PCR reaction solution and 5 μL of enzyme solution are taken to prepare 20 μL of qualitative reaction liquid; in the step S03, 10 μL of the extraction product, wild-type positive control, drug-resistant positive control, and negative control obtained in step S01 are taken respectively, and 20 μL of PCR qualitative reaction liquid obtained in the above step S02 are added respectively to prepare a total of 30 μL of PCR system.

[0024] Compared with the existing technology, the present invention uses PCR technology combined with high-resolution melting curve analysis technology to develop a detection kit for respiratory infection pertussis nucleic acid and drug resistance gene sites; only one set of primers and probes is used to diagnose pertussis infection in the early stage, and at the same time make an accurate judgment on the drug resistance of Bordetella pertussis, providing a reliable experimental means for the prevention and control of pertussis infection in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0026] Figure 1 This is the melting curve of Example 1.

[0027] Figure 2This is the melting curve of Control Example 1.

[0028] Figure 3 This is the melting curve of control example 2. Figure 4 Validation of the kit for clinical samples. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0030] Embodiment 1:

[0031] A kit for detecting pertussis nucleic acid and drug-resistant gene by using high-resolution melting curve comprises a fluorescent PCR reaction solution, an enzyme solution, a wild-type positive control substance, a drug-resistant positive control substance and a negative control substance. The fluorescent PCR reaction solution mainly contains 20 mM MgCl2, 25 mM dNTPs, an upstream primer corresponding to a pertussis target, a downstream primer corresponding to a pertussis target and a fluorescent probe corresponding to the pertussis target. The concentrations of the upstream primer corresponding to the pertussis target, the downstream primer corresponding to the pertussis target and the fluorescent probe corresponding to the pertussis target are 500 nM, 100 nM and 500 nM respectively, the enzyme amount is 8 U, the pertussis target is site 2047 of the 23S rRNA gene of Bordetella pertussis; the sequence of the upstream primer is shown in SEQ ID NO: 1; the sequence of the downstream primer is shown in SEQ ID NO: 2; the sequence of the fluorescent probe is shown in SEQ ID NO: 3, and specifically, as shown in Table 1.

[0032] Table 1

[0033]

[0034] In Table 1, the A base after the "+" in the fluorescent probe sequence is a locked nucleic acid.

[0035] The positive controls in this kit include wild-type positive controls and drug-resistant positive controls, which are dissolved in TE buffer to 10 6 copies / ml, wherein the wild-type positive control target amplification sequence is shown in SEQ ID NO: 4, and the drug-resistant positive control target amplification sequence is shown in SEQ ID NO: 5, specifically, as shown in Table 2.

[0036] Table 2

[0037]

[0038] The negative control in this kit is sterile water for injection, and the enzyme solution contains Taq DNA polymerase. This kit is stored at -20℃, and repeated freezing and thawing should be minimized.

[0039] The test kit also includes a box body and a box cover. The box body is provided with eight container holes for placing two fluorescent PCR reaction solution tubes, two enzyme solution tubes, two positive control tubes, and two negative control tubes. The fluorescent PCR reaction solution tube contains fluorescent PCR reaction solution, the enzyme solution tube contains enzyme solution, the positive control tube contains positive control substance, and the negative control tube contains negative control substance. The eight container holes can be used to record the fluorescent PCR reaction solution tube placement hole, enzyme solution tube placement hole, wild-type positive control tube placement hole, drug-resistant positive control tube placement hole, and negative control tube placement hole.

[0040] Embodiment 2:

[0041] A method for detecting the 2047 site of the 23S rRNA gene of Bordetella pertussis using the above kit, wherein a wild-type positive control, a drug-resistant positive control and a negative control should be set up for each detection, comprising the following steps:

[0042] (1) Nucleic acid extraction: Take 0.5 mL of throat swab sample from a child suspected of pertussis infection in a clinical setting and place it in an EP tube. Use magnetic bead method or column extraction method to extract nucleic acid from the throat swab of the sample to be tested.

[0043] (2) Reagent preparation: Take 15 μL of PCR reaction solution and 5 μL of enzyme solution to prepare 20 μL of qualitative reaction liquid;

[0044] (3) Amplification detection: Take 10 μL of the extraction product obtained in step S01, 10 μL of the wild-type positive control, 10 μL of the drug-resistant positive control or 10 μL of the negative control, respectively, and add 20 μL of the qualitative reaction liquid obtained in the above step S02, and prepare a PCR system of 30 μL in total. The detection is carried out on a fluorescent quantitative PCR instrument with a single color or above. The specific reaction procedure is (taking the domestically produced Hongshi SLAN-96P fluorescent quantitative PCR instrument as an example), as shown in Table 3.

[0045] Table 3 Reaction program settings SLAN-96P

[0046]

[0047] (4) Melting curve analysis

[0048] The test results are analyzed as follows Figure 1 shown.

[0049] When the peak temperature of the melting curve of the FAM fluorescence channel is 55.2±0.5℃, the 2047 position of the 23SrRNA gene of B. pertussis has not mutated, and it is judged as positive for B. pertussis nucleic acid;

[0050] When the peak temperature of the melting curve of the FAM fluorescence channel is 44.7±0.5℃, the 2047 position of the 23SrRNA gene of Bordetella pertussis is mutated; it is judged as positive for antibiotic-resistant Bordetella pertussis nucleic acid;

[0051] When no melting curve peak appears in the FAM fluorescence channel, it is judged as pertussis negative.

[0052] Embodiment 3:

[0053] Kit Validation for Clinical Samples

[0054] Common clinical Bordetella pertussis positive samples were selected, DNA was extracted, and then the kit of the present invention was used for verification.

[0055] The selected samples are positive samples of bacteria such as Bordetella pertussis, Bordetella parapertussis, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus agalactiae, Enterococcus, Staphylococcus epidermidis, Acinetobacter baumannii, Pseudomonas aeruginosa, and cytomegalovirus, Epstein-Barr virus, influenza virus, Chlamydia trachomatis, and human genome, and the results show that the reagent of the present invention can correctly detect Bordetella pertussis and can correctly distinguish the mutation of the 23S rRNA gene 2047 site. The positive samples of bacteria such as Bordetella parapertussis, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus agalactiae, Enterococcus, Staphylococcus epidermidis, Acinetobacter baumannii, Pseudomonas aeruginosa, and cytomegalovirus, Epstein-Barr virus, influenza virus, Chlamydia trachomatis, and human genome are all negative, indicating that the detection specificity of the present invention is good. By testing the known concentration of plasmid, the wild type and mutant types of Bordetella pertussis were detected at a concentration of 1000 copies / ml, and the positive rate was 95%. Figure 4 , indicating that the detection sensitivity of the present invention is excellent.

[0056] The present invention uses a single reaction tube to detect the high-resolution melting curve of the FAM fluorescence channel, and realizes the early diagnosis of wild-type and drug-resistant types of Bordetella pertussis by accurately detecting the 2047 site of the 23SrRNA gene. It only requires the cost of a reaction reagent containing a set of primer probes, and the detection reaction can be performed on a general fluorescent quantitative PCR instrument. The result interpretation is simple, and there is no risk of contamination without opening the cover. It can be suitable for medical institutions with ordinary clinical PCR laboratories.

[0057] The present invention uses MGB quenching group combined with locked nucleic acid base technology, and adopts the technical principle of PCR combined with melting curve to develop a detection kit for detecting pertussis nucleic acid and drug resistance genes. Because the locked nucleic acid base highly enhances the recognition of the variant site, and the MGB chemical group promotes the binding strength of the probe, the judgment result is clearer; the invention has the sensitivity and specificity of fluorescent quantitative PCR, and can diagnose pertussis nucleic acid and drug resistance at the early stage at the same time, with simpler operation and lower cost. It provides a reliable experimental means for the diagnosis of pertussis infection in clinical practice.

[0058] Comparative Example 1:

[0059] The kit was prepared in the same manner as in Example 1 and Example 2, with only the fluorescent probe being changed to study the effect of the fluorescent probe on the detection sensitivity and accuracy. The primer sequences and fluorescent probes are shown in Table 4:

[0060] Table 4

[0061]

[0062] The difference between Table 4 and Table 1 is that the fluorescent probe is a conventional fluorescent probe, and no locked nucleic acid is added. The melting curve results are as follows Figure 2 shown.

[0063] Comparative Example 2:

[0064] The kit was prepared in the same manner as in Example 1 and Example 2, with only the fluorescent probe being changed to study the effect of the fluorescent probe on the detection sensitivity and accuracy. The primer sequences and fluorescent probes are shown in Table 5:

[0065] Table 5

[0066]

[0067] In Table 5, the A base after the "+" in the fluorescent probe sequence is a locked nucleic acid.

[0068] The difference between Table 5 and Table 1 is that the fluorescent probe quenching group is BHQ1, and the melting curve results are as follows Figure 3 shown.

[0069] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A kit for detecting pertussis nucleic acid and drug resistance gene by high-resolution melting curve, characterized in that: The invention comprises an upstream primer, a downstream primer and a fluorescent probe, wherein the sequence of the upstream primer is: 5′-TTCCTTGTCGGGTAAGTTCCG-3′; the sequence of the downstream primer is: 5′-CCTATCCTACACAGGCCGGTTC-3′; the fluorescent probe is: FAM-ACGGAAAGACCC-M GB; wherein the fifth base A in the nucleic acid sequence of the fluorescent probe is a locked nucleic acid modified base.

2. The kit for detecting pertussis nucleic acid and drug resistance gene by high resolution melting curve according to claim 1, characterized in that: The kit comprises a fluorescent PCR reaction solution, an enzyme solution, a wild-type Bordetella pertussis positive control substance, a drug-resistant Bordetella pertussis positive control substance, and a negative control substance; wherein the fluorescent PCR reaction solution contains a PCR buffer, MgCl2, dNTPs, the upstream primer, the downstream primer, and the fluorescent probe.

3. The kit for detecting pertussis nucleic acid and drug resistance gene by high resolution melting curve according to claim 2, characterized in that: The kit also includes a box body, which is provided with a plurality of container holes, and the plurality of container holes are used to respectively place fluorescent PCR reaction solution tubes, enzyme solution tubes, wild-type positive control substances, drug-resistant positive control substances, and negative control substance tubes.

4. The kit for detecting pertussis nucleic acid and drug resistance gene by high resolution melting curve according to claim 2, characterized in that: The detection target sequence of the kit is wild-type Bordetella pertussis and / or drug-resistant Bordetella pertussis; the detection target sequence of the wild-type Bordetella pertussis is shown in SEQ ID NO: 4, and the detection target sequence of the drug-resistant Bordetella pertussis is shown in SEQ ID NO:

5.

5. A kit for detecting pertussis nucleic acid and drug resistance gene by high resolution melting curve according to any one of claims 2 to 4, characterized in that: The enzyme solution contains Taq DNA polymerase.

6. A kit for detecting pertussis nucleic acid and drug resistance gene by high resolution melting curve according to any one of claims 2 to 4, characterized in that: The negative control substance is sterile water.

7. A kit for detecting pertussis nucleic acid and drug resistance gene by high resolution melting curve according to any one of claims 2 to 4, characterized in that: The sample to be tested is a throat swab or a nasal swab.

8. The method for using the kit for detecting pertussis nucleic acid and drug resistance gene by high resolution melting curve according to claim 2, characterized in that: The following steps are involved: Step S01, nucleic acid extraction: extracting nucleic acid from the sample to be tested using a magnetic bead method or a column extraction method; Step S02, reagent preparation: mixing the PCR reaction solution and the enzyme solution to prepare a PCR qualitative reaction liquid; Step S03, amplification detection: taking the nucleic acid extraction product obtained in step S01, the wild-type Bordetella pertussis positive control substance, the drug-resistant Bordetella pertussis positive control substance, and the negative control substance, respectively adding them to the PCR qualitative reaction liquid obtained in the above step S02, preparing a PCR system, and performing detection on a single-color fluorescence quantitative PCR instrument; Step S04, performing fluorescence quantitative result analysis.

9. The method for using the kit for detecting pertussis nucleic acid and drug resistance gene by high resolution melting curve according to claim 8, characterized in that: When the peak temperature of the melting curve of the FAM fluorescence channel is 55.2±0.5℃, the 2047 position of the 23SrRNA gene of B. pertussis has not mutated, and it is judged as positive for B. pertussis nucleic acid; When the peak temperature of the melting curve of the FAM fluorescence channel is 44.7±0.5℃, the 2047 position of the 23SrRNA gene of Bordetella pertussis is mutated; it is judged as positive for antibiotic-resistant Bordetella pertussis nucleic acid; When no melting curve peak appears in the FAM fluorescence channel, it is judged as pertussis negative.

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