Kit for detection of polymorphisms of Helicobacter pylori drug resistance gene by multiplex fluorescent PCR melting curve method

Abstract

The invention discloses a kit for detecting the polymorphism of the drug-resistant gene of Helicobacter pylori by a multiplex fluorescence PCR melting curve method. The kit includes nucleic acid extraction reagents and nucleic acid amplification reagents; nucleic acid extraction reagents include superparamagnetic silica nano-magnetic beads, lysate, washing liquid, and eluent; nucleic acid amplification reagents include: Primer pairs and probes corresponding to rRNA gene, 16S rRNA gene, gyr A gene, internal standard gene human housekeeping gene β‑globin. The invention can simultaneously detect three drug-resistant loci and one internal standard gene in a single tube, improve the throughput of sample detection; meanwhile, improve the interference between multiple pairs of primers and probes in the detection system, and effectively improve the sensitivity and specificity of reagent detection sex.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a kit for detecting the polymorphism of the drug-resistant gene of Helicobacter pylori by a multiplex fluorescent PCR melting curve method. This kit is used for the qualitative detection of common drug-resistant mutations in the Helicobacter pylori gyrA gene, 23S rRNA gene, and 16S rRNA gene in gastric mucosal tissue samples, and assists in the selection of drugs for clinical treatment. Background technique [0002] Current detection methods for drug resistance to Helicobacter pylori include drug susceptibility testing, nucleic acid sequencing, and TaqMan real-time fluorescent PCR. Drug susceptibility testing is the gold standard method for the detection of drug resistance of Helicobacter pylori, which can intuitively determine which antibiotics Helicobacter pylori is resistant to; nucleic acid sequencing can use different sequencing Nucleic acid sequences are compare...

AI Technical Summary

Problems solved by technology

There are the following problems in the detection process: 1. The primers modified by Blocker compete with the amplification primers to bind to the target, reducing the binding efficiency of the amplification primers to the target, resulting in a decrease in sensitivity; 2. The detection principle of ARMS primers relies on individual bases Therefore, there will be false positive results caused by primer mismatch, resulting in a decrease in specificity; 3. In this system, different ARMS primers and blocker primers need to be set for different mutation sites, and the cost of reagents will increase. Relatively high, increasing the cost of testing and increasing the cost of testing for patients

4. To detect the 7 base mutation directions of a sample, 1 sample needs to be tested 8 times to determine whether the base has mutated, resulting in cumbersome operations, low detection throughput, and high detection costs in clinical testing, which is not suitable for clinical use.

However, it has the following problems: 1. Using the fluorescent PCR method to detect mutations in Helicobacter pylori, only one mutation site can be detected in a single direction in a single well, and different sites and different mutation directions need to be detected in different positions. It is carried out in a reaction tube, and the detection throughput of the sample is small; 2. This patent can only detect whether the 2142 and 2143 sites of the 23S rRNA gene of Helicobacter pylori are mutated, and cannot determine whether the sample is infected with wild-type Helicobacter pylori; 3. This patent can only detect mutations at sites 2142 and 2143 on the 23S rRNA gene of Helicobacter pylori, and cannot detect mutations in quinolone antibiotics and tetracycline resistance genes at the same time, increasing the cost and time of clinical testing

4. The kit described in this patent does not have an internal control, and cannot monitor whether there is any abnormality in the sample during the process of sampling, extraction, amplification, etc., and cannot guarantee the accuracy of the results

[0007] Chinese invention patent application CN201911327235 discloses molecular beacon probes, kits and detection methods for detecting Helicobacter pylori. This patent uses beacon probes for fluorescence in situ hybridization detection, and there are several problems as follows: 1. The detection process steps are comparatively It is cumbersome, after hybridization, multiple cleaning and other steps, the operation time is long, and the signal is easily lost during the operation, resulting in false negative results; 2. The probe cannot hybridize to the target fragment 100%, and the hybridization rate is affected by the probe , reduce the detection sensitivity; 3. In the gastric mucosa sample, the method of probe hybridization is used to detect Helicobacter pylori without PCR amplification.

4. Although this method does not need to be equipped with a PCR instrument, the late hybridization results need to be interpreted by visual inspection under a microscope, the results are not preserved completely, and the result judgment is greatly affected by human factors, and the use of microscopes limits the use of this method. This detection method cannot be extended to clinical units Or basic medical units to conduct relevant tests

Due to the non-specific binding of fluorescent dyes, the number of targets detected in a single reaction tube is limited, so there are certain technical difficulties or obstacles in the detection of multiple drug resistance genes of Helicobacter pylori by fluorescence melting curve method

At present, there is no literature disclosing the application of melting curve method to detect multiple drug resistance genes of Helicobacter pylori

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