Bridging primer probe combination, kit and detection method for fluorescent quantitative PCR detection of Helicobacter pylori point mutations

Abstract

The present invention discloses a bridge primer-probe combination, a kit, and a detection method for detecting Helicobacter pylori point mutations using fluorescent quantitative PCR. The detection method provided by the present invention simplifies the optimization process of the multiplex PCR system and overcomes the shortcomings of the traditional E-Test experimental research method for bacterial resistance, such as the long isolation and cultivation period and high difficulty of the culture. The method provided by the present invention takes about two hours to detect, greatly improving detection efficiency. Compared with conventional multiplex PCR, it has higher sensitivity, with a detectable sensitivity of up to 500 copies / ml. It also has strong specificity and reduces nonspecific amplification of primers.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a bridge primer-probe combination, a kit and a detection method for detecting Helicobacter pylori point mutations by fluorescent quantitative PCR. Background Art

[0002] Helicobacter pylori (Hp) is the direct cause of chronic active gastritis, closely associated with peptic ulcers and mucosa-associated lymphoid tissue lymphoma, and a risk factor for gastric cancer. Eradication of H. pylori is a key clinical strategy for the treatment of chronic gastritis. It not only accelerates the healing of peptic ulcers but also significantly reduces ulcer recurrence and postoperative recurrence of early-stage gastric cancer. Currently, clarithromycin is one of the most commonly used antibiotics for eradication of H. pylori in clinical practice. However, with the widespread use of clarithromycin and other antibiotics, the prevalence of H. pylori resistance has been increasing annually.

[0003] The traditional method for studying bacterial resistance is the E-Test experiment, but Helicobacter pylori is a microaerophilic bacterium, and the isolation and culture cycle of the strain is long, requiring about a week. In addition, the culture is difficult and the success rate is low, which cannot provide doctors with a basis for diagnosis and treatment in the first place.

[0004] It is now generally understood that the mechanism of clarithromycin resistance in Helicobacter pylori is due to point mutations in the V region of the 23S rRNA gene in clarithromycin-resistant H. pylori. These mutations alter ribosome conformation and the macrolide binding site, which in turn weakens H. pylori's affinity for macrolides, preventing the drug from inhibiting bacterial protein synthesis and ultimately leading to drug resistance. The mutation rates of A2142C, A2142G, and A2143G in the 23S rRNA gene of clarithromycin-resistant H. pylori are 1.5%, 6.2%, and 84.6%, respectively. Therefore, the use of PCR to detect mutations in the H. pylori 23S rRNA gene has gained some recognition and is often used as a basis for research on clarithromycin resistance in H. pylori.

[0005] The publicly disclosed patented technology "A kit for detecting drug-resistant gene polymorphisms of Helicobacter pylori by multiplex fluorescence PCR melting curve method" (publication number: CN111850154A) provides a kit for the A2142C and A2142G sites in the 23rRNA gene. However, the melting curve is a mathematical deduction after all, and the error of the instrument is also included in the result, and the requirements for the amplification instrument are also high. In addition, non-specific binding of primers will occur when performing multi-target specific amplification in the same reaction system. The patent "A kit, a method for using the kit, and the purpose of the kit" (publication number: CN107312832A) provides a specific primer pair for detecting the A2142G / C mutation of the 23S rRNA gene. It uses the ARMS-PCR principle for detection, and the conditions are difficult to explore. Summary of the Invention

[0006] In order to overcome the problems of high requirements for amplification instruments and poor specificity in the existing detection technology, the present invention provides a Qiaolian primer-probe combination for fluorescent quantitative PCR detection of Helicobacter pylori point mutations, and also provides a kit and a detection method for detecting the point mutation A2142G at the 2142 polymorphic site of the Helicobacter pylori 23S rRNA gene. The bridging primer consists of three parts: a 5' end part, a 3' end part, and a polyxanthine connecting the two ends.

[0007] The first object of the present invention is to provide a bridge primer-probe combination for detecting Helicobacter pylori point mutations by fluorescent quantitative PCR, comprising:

[0008] As shown in SEQ ID No. 1, the base 1 is inserted between the base sequence GA of the upstream primer HP-A2142G-DPOF, and its base sequence is: CTACCCGCGGCAAGACGGG (I) n AAGACCC;

[0009] As shown in SEQ ID No. 2, the downstream primer HP-A2142G-DPOR has one base inserted between the base sequences AG, and its base sequence is: TACTTCAA (I) n AGCCTCCCACCTAT;

[0010] As shown in SEQ ID No. 3, the probe HP-A2142G-DPOP has a base sequence of: FAM-ACCCCGTGGACCTTTACTACAACT-BHQ1;

[0011] Wherein, I represents hypoxanthine, and n represents the number of hypoxanthines, which is selected from 3-8, preferably 5 or 6.

[0012] The second object of the present invention is to provide a kit for detecting Helicobacter pylori point mutations by fluorescent quantitative PCR, comprising the bridge primer-probe combination for detecting Helicobacter pylori point mutations by fluorescent quantitative PCR.

[0013] Preferably, the kit further comprises a reaction solution, an enzyme mixture, a positive control, and a negative control; the reaction solution comprises dNTPs, Mg 2+ , Tris-HCl, and the enzyme mixture includes reverse transcriptase and Taq DNA polymerase.

[0014] The kit provided by the present invention can be used to detect the Helicobacter pylori 23S rRNA gene point mutation A2142G in gastric mucosal tissue samples or fecal samples of patients infected with Helicobacter pylori in vitro.

[0015] As a third object of the present invention, it is to provide a method for detecting the Helicobacter pylori 23S rRNA gene point mutation A2142G, comprising the following steps:

[0016] Step (1), extracting nucleic acid from the sample to be tested;

[0017] Step (2), performing a fluorescent quantitative PCR amplification reaction using the nucleic acid extracted in step (1) as a template;

[0018] Step (3), analyzing the results based on the FAM channel amplification curve and CT value.

[0019] Furthermore, the PCR reaction system in step (2) is calculated as follows based on 25 μl:

[0020]

[0021] Furthermore, the reaction conditions of the fluorescence quantitative PCR in step (2) are: 94°C for 5 minutes; 94°C for 15 seconds, 60°C for 30 seconds, collecting FAM channel fluorescence, for a total of 40 cycles.

[0022] Furthermore, in step (3), the judgment criteria for the test results are: if the CT value is ≤38 and there is an S-shaped amplification curve, it is positive for the Helicobacter pylori 23S rRNA gene A2142G point mutation; if the CT value is >38 or there is no CT value, it is negative for the Helicobacter pylori 23S rRNA gene A2142G point mutation.

[0023] The fourth object of the present invention is to provide the use of the bridge primer-probe combination in detecting point mutations in Helicobacter pylori, and the use of the kit in detecting point mutations in Helicobacter pylori.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention provides a method for detecting Helicobacter pylori point mutations using fluorescent quantitative PCR. This method simplifies the optimization process of the multiplex PCR system and overcomes the shortcomings of traditional bacterial resistance E-Test experimental research methods, such as the long isolation and culture period and the difficulty of culture. The method provided by the present invention takes less than two hours to detect, greatly improving detection efficiency.

[0026] 2. The detection method provided by the present invention has higher sensitivity than conventional multiplex PCR, with a detectable sensitivity of up to 500 copies / ml; it has strong specificity and reduces non-specific amplification of primers.

[0027] 3. Compared with the melting curve method, this application adopts the bridge primer technology, which avoids the result error of the melting curve and the non-specific binding of primers in multiplex PCR, and is suitable for the detection of point mutation sequences.

[0028] 4. At the same time, the bridge primer method used in this application is simpler and more convenient to operate than the ARMS-PCR principle, reduces the non-specific amplification of primers, and has higher sensitivity compared with it. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0030] Figure 1 The diagram shows the amplification effect at different primer concentrations.

[0031] Figure 2 This is a graph showing the detection results in a specificity experiment.

[0032] Figure 3 This is the test result diagram of sensitivity evaluation experiment. DETAILED DESCRIPTION

[0033] In order to more clearly describe the technical solution of the present invention, the following is further described in conjunction with specific embodiments, but it is not intended to limit the present invention. These are only some embodiments of the present invention.

[0034] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0035] Example 1:

[0036] 1. Materials and Methods

[0037] 1.1 Materials and Equipment

[0038] Detection reagents: The nucleic acid extraction kit (magnetic bead method) of Shandong Amber Biotechnology Co., Ltd. was used, the reaction solution and enzyme mixture were purchased from Zhuhai Baorui Biotechnology Co., Ltd., the primer probe was synthesized by Shanghai Bailige Biotechnology Co., Ltd., and the experimental samples were gastric mucosal tissue samples from patients undergoing gastroscopy in the hospital.

[0039] Main equipment: fully automatic nucleic acid extraction instrument AP-9601 from Shandong Amber Biotechnology Co., Ltd., and fully automatic PCR analysis system from Xi'an Tianlong Technology Co., Ltd.

[0040] 1.2 Experimental methods

[0041] 1.2.1 Nucleic acid extraction

[0042] Use sterile gastroscopic forceps to obtain gastric mucosal tissue samples, add 180 μl of D-PBS buffer and 30 μl of proteinase K, and shake to dissolve and digest the samples for later use.

[0043] Nucleic acid extraction was performed on the experimental samples according to the instructions for the Nucleic Acid Extraction Kit (Magnetic Bead Method) from Shandong Amber Biotechnology Co., Ltd. 200 μl of the processed sample was added to the wells of the acid extraction kit's lysate plate. 20 μl of Proteinase K solution was then added. The reagents were then placed in the nucleic acid extraction instrument in the correct order. The AP91005 program was selected and run. At the end of the run, the extracted nucleic acids were located in the corresponding wells of the eluate plate. The nucleic acids were then transferred to a clean centrifuge tube for later use.

[0044] 1.2.2 Primer and probe design

[0045] Referring to the design method and requirements of bridge primers, a primer probe was designed based on the A2142G point mutation at the 2142 polymorphic site of the Helicobacter pylori 23S rRNA gene. The sequence is as follows:

[0046] As shown in SEQ ID No. 1, five I bases are inserted between the base sequence GA of the upstream primer HP-A2142G-DPOF, and its base sequence is: CTACCCGCGGCAAGACGGGIIIIIAAGACCC;

[0047] As shown in SEQ ID No. 2, 5 I bases are inserted between the base sequence AG of the downstream primer HP-A2142G-DPOR, and its base sequence is: TACTTCAAIIIIIAGCCTCCCACCTAT;

[0048] As shown in SEQ ID No. 3, the base sequence of the probe HP-A2142G-DPOP is FAM-ACCCCGTGGACCTTTACTACAACT-BHQ1.

[0049] 1.2.3 Reaction system

[0050]

[0051] The reaction solution is a mixture of 80% DEPC water, 10% Tris-Cl, 10% KCl and MgCl2, and the enzyme mixture is 90% DEPC water and 10% Taq enzyme.

[0052] The reaction conditions were: 94°C for 5 minutes; 94°C for 15 seconds, and 60°C for 30 seconds (collecting FAM channel fluorescence), for a total of 40 cycles.

[0053] 1.2.4 Specificity evaluation

[0054] Evaluate the specificity of the established reaction system using the reaction system described in 1.2.3, using nucleic acids from four non-H. pylori samples and four H. pylori samples with non-target point mutations extracted in 1.2.1 as templates, a sample with the A2142G point mutation (AAGACGGGA) as a positive control, and water as a negative control.

[0055] 1.2.5 Sensitivity evaluation

[0056] E. coli cell samples transfected with a specific Helicobacter pylori 23S rRNA gene fragment containing the A2142 point mutation were used as reference samples for the detection limit. These samples were diluted to five template concentrations: 5000 copies / ml, 2000 copies / ml, 1000 copies / ml, 500 copies / ml, and 200 copies / ml. 5 μl of each sample was used as a template for sensitivity testing according to the method in 1.2.3.

[0057] 1.2.6 Annealing temperature sensitivity experiment

[0058] According to the reaction system in 1.2.3, set the annealing temperature in the reaction conditions to 45°C-65°C, with 5°C as one step and a total of 5 steps, and perform fluorescence quantitative PCR experiment to analyze the results.

[0059] 2. Results Analysis

[0060] 2.1 Establishment of detection method:

[0061] By changing and comparing the reaction system, it was determined that the amplification effect was best when the final concentration of upstream and downstream primers was 0.3μM, and the amplification effect was best when the final concentration of probe was 0.1μM. Figure 1 shown.

[0062] 2.2 Specificity evaluation

[0063] The nucleic acid template test results of 4 non-Helicobacter pylori samples and 4 Helicobacter pylori samples without the target point mutation were negative, and the test results of the Helicobacter pylori sample with the A2142G point mutation were positive. Figure 2 The results showed that the established detection method has good specificity and can be used to detect the point mutation A2142G at the 2142 polymorphic site of the 23S rRNA gene of Helicobacter pylori.

[0064] 2.3 Sensitivity evaluation

[0065] Test results such as Figure 3 The results showed that the CT value of the sample with a concentration of 500 copies / ml was 36-37, and the detection rate was 100% after 20 repeated tests on the sample with this concentration. The sample with a concentration of 50 copies / ml had no detection results, indicating that the detection sensitivity of the present invention reached 500 copies / ml. In addition, the CT value of the sample with a concentration of 200 copies / ml was 37-39, and the detection rate was 80% after 20 repeated tests on the sample with this concentration. It can be seen that the detection sensitivity of this method is high.

[0066] 2.4 Annealing temperature sensitivity experiment

[0067] In the annealing temperature sensitivity experiment of the present invention, the annealing temperature was set to five gradients of 45°C-65°C, and the best amplification effect was achieved when the annealing temperature was 60°C.

[0068] Example 2:

[0069] Six sets of primers (with 3-8 I bases) were designed according to the target gene sequence of Helicobacter pylori. Nucleic acid amplification experiments were performed 10 times to detect weak positive quality control products. According to the average CT value of the reaction, it was concluded that the best effect was achieved when the I base was 5 or 6.

[0070]

[0071]

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical aspects of the present invention and are not intended to limit the scope of protection of the present invention. 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 essence and scope of the technical solutions of the present invention. Sequence Listing <110> Shandong Amber Biotechnology Co., Ltd. <120> Bridging primer probe combination, kit and detection method for fluorescent quantitative PCR detection of Helicobacter pylori point mutations <130> 2022.06.10 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 26 <212> DNA <213> HP-A2142G-DPOF <400> 1 ctacccgcgg caagacggga agaccc 26 <210> 2 <211> 22 <212> DNA <213> HP-A2142G-DPOR <400> 2 tacttcaaag cctcccacct at 22 <210> 3 <211> 24 <212> DNA <213> HP-A2142G-DPOP <400> 3 accccgtgga cctttactac aact 24

Claims

1. A bridging primer-probe combination for detecting Helicobacter pylori point mutations by fluorescent quantitative PCR, characterized in that: include: As shown in SEQ ID No. 1, the base 1 is inserted between the base sequence GA of the upstream primer HP-A2142G-DPOF, and its base sequence is: CTACCCGCGGCAAGACGGG (I) n AAGACCC; As shown in SEQ ID No. 2, the downstream primer HP-A2142G-DPOR has one base inserted between the base sequence AA, and its base sequence is: TACTTCAA (I) n AGCCTCCCACCTAT; The probe HP-A2142G-DPOP is shown in SEQ ID No. 3; Wherein, I represents hypoxanthine, and n is the number of hypoxanthines; 5 or 6 I bases are inserted into the upstream primer and the downstream primer, respectively.

2. A kit for detecting Helicobacter pylori point mutations by fluorescent quantitative PCR, characterized in that: The invention comprises the bridge primer-probe combination for detecting Helicobacter pylori point mutations by fluorescent quantitative PCR according to claim 1.

3. A kit for detecting Helicobacter pylori point mutations by fluorescent quantitative PCR according to claim 2, characterized in that: The kit also includes a reaction solution, an enzyme mixture, a positive control, and a negative control; the reaction solution includes dNTPs, Mg 2+ , Tris-HCl, and the enzyme mixture includes reverse transcriptase and Taq DNA polymerase.

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