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Primer and probe set for detecting helicobacter pylori drug-resistant mutation sites and application thereof

A drug-resistant mutation site, Helicobacter pylori technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve the problems of low sensitivity, time-consuming, complicated operation, etc., and achieve high sensitivity , short time-consuming effect

Pending Publication Date: 2020-01-14
浙江默乐生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Helicobacter pylori is a helical, slightly anaerobic, Gram-negative bacillus that requires very harsh growth conditions. Therefore, the cultivation of Helicobacter pylori is difficult, complicated, and time-consuming (the whole process of drug susceptibility testing is usually takes 1-2 weeks)
Therefore, the drug susceptibility test of Helicobacter pylori is costly and has low sensitivity

Method used

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  • Primer and probe set for detecting helicobacter pylori drug-resistant mutation sites and application thereof
  • Primer and probe set for detecting helicobacter pylori drug-resistant mutation sites and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, primer, probe design

[0051] By analyzing the sequences of Helicobacter pylori amoxicillin resistance gene PBP1A and tetracycline resistance gene 16SrRNA, drug resistance mutation sites were selected, and specific primer probes were designed, as shown in Table 1 below.

[0052] Table 1

[0053] SEQ ID NO.1 PBP-F1 aacaaaaccacgcatggcaccccaca SEQ ID NO.2 PBP-F2 aaaaccgggacttctaacaaat SEQ ID NO.3 PBP-F3 tttagaaatcgccggtaaatg SEQ ID NO.4 PBP-block1 agtgaacaaaaccacgcatggcaccccagc SEQ ID NO.5 PBP-block2 tcgccggtaaaaccgggacttctaacaaca SEQ ID NO.6 PBP-block3 cattaaaggtttagaaatcgccggtaaaac SEQ ID NO.7 PBP-R gcgttcctcgctatcgtctg SEQ ID NO.8 PBP-P acgatttctttacgcaagcctttggggacatc SEQ ID NO.9 16S-F agcatgtggtttaattcgattc SEQ ID NO.11 16S-block agcggtggagcatgtggtttaattcgaaga SEQ ID NO.10 16S-R cgctcgttgcggggactta SEQ ID NO.12 16S-P ccaacatctcacgacacgagctgacga...

Embodiment 2

[0058] Embodiment 2, fluorescent PCR

[0059] The primers, blocking sequences and probes provided in Example 1 can be used to perform fluorescent PCR to detect drug resistance mutation sites of Helicobacter pylori.

[0060] Table 2 shows the formula of PCR reaction solution.

[0061] Table 2

[0062] Reagent name Volume (μL) 10×PCR Buffer (Mg 2+ Plus)

2.5 MgCl 2 (1M)

0.05 dNTPs [contains dUTP] (25mM) 0.2 PBP-F1 0.6 PBP-F2 0.6 PBP-F3 0.6 PBP-block1 1.2 PBP-block2 1.2 PBP-block3 1.2 PBP-R 0.6 PBP-P 0.3 16S-F 0.6 16S-block 1.2 16S-R 0.6 16S-P 0.3 HS Taq (5U) 0.4 UNG enzyme (1U) 0.1 Sterile RNase-free water 7.75 template 5 total 25

[0063] Wherein, in the prepared reaction solution, the concentration of each blocking sequence is twice the concentration of the respective corresponding primers. The optimal concentration of each primer was...

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Abstract

The invention relates to the technical field of digestive tract related disease diagnosis, in particular to a primer and a probe set for detecting helicobacter pylori drug-resistant mutation and an application thereof. The primer and probe set comprise a first PCR detection primer pair for detecting amoxicillin resistance mutation site of a helicobacter pylori PBP1A gene, wherein the first PCR detection primer pair consists of a first upstream primer and a first downstream primer, and the first upstream primer is any one or any two of the upstream primers of the upstream primer used for detecting the Ser414Ag mutation of the PBP1A gene, the upstream primer used to detect the Thr556 Ser mutation of the PBP1A gene, and the upstream primer used to detect the Asn562 Tyr mutation of the PBP1A gene, or a combination of three upstream primers; and a first probe; and at least one blocking sequence comprising a blocking sequence corresponding to the first upstream primer.

Description

technical field [0001] The invention relates to the technical field of diagnosis of digestive tract-related diseases, in particular to primers and probe sets and applications for detecting drug-resistant mutation sites of Helicobacter pylori. Background technique [0002] Helicobacter pylori (Hp) is the most widespread pathogen in the world, about 50% of the world's people are infected with Helicobacter pylori, and in my country, 40%-70% of the population is infected with Helicobacter pylori. Helicobacter pylori mainly causes diseases related to the digestive system of the gastrointestinal tract, such as chronic gastritis, gastric ulcer, duodenal ulcer, gastric cancer and so on. At present, the treatment of Helicobacter pylori mainly adopts triple or quadruple therapy containing antibiotics, and with the extensive use of antibiotics, Helicobacter pylori has become resistant to antibiotics. Therefore, the detection of drug resistance of Helicobacter pylori is of great signif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12N15/11
CPCC12Q1/689C12Q1/686C12Q2600/156C12Q2600/106C12Q2563/107
Inventor 吕棠山李杨霞王伟伟贾俊玲曾丽杨淼
Owner 浙江默乐生物科技有限公司
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