LAMP primer group for detecting helicobacter pylori cytotoxin related protein cagA and application thereof

A Helicobacter pylori and cytotoxin technology, applied in the field of biology, can solve the problems of long detection time, insufficient sensitivity, and no symptoms, and achieve the effect of simple operation, high sensitivity, and strong specificity

Inactive Publication Date: 2018-09-14
厦门蓝特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In addition, HP typing is difficult because it may lie in the host for a long time without showing any symptoms
At present, the most widely used detection methods are: histopathological examination, C13 / C14 half-life examination, serological detection and PCR reaction, etc. These methods have high requirements for instruments, long detection time, insufficient sensitivity and difficult strain typing

Method used

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  • LAMP primer group for detecting helicobacter pylori cytotoxin related protein cagA and application thereof
  • LAMP primer group for detecting helicobacter pylori cytotoxin related protein cagA and application thereof
  • LAMP primer group for detecting helicobacter pylori cytotoxin related protein cagA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cytotoxin-associated protein in Helicobacter pylori ( cagA ) detection limit test

[0034] 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL of HP strain ATCC 700392 DNA were used as templates, respectively.

[0035] LAMP reaction system includes: 1×ThermoPol reaction buffer; 6 mM MgSO4; 0.8 M betaine; 1.4mM dNTPs; the above three pairs of primers, 0.2 μM F3 / B3; 1.6 μM FIP / BIP; 0.8 μM LF / LB; Bst DNA polymerase 320 U / mL; DNA template 1 μL, make up to 20 μL with enzyme-free water. For the blank control group, no DNA was added, and 1 μL of enzyme-free water was added accordingly.

[0036] React in a water bath at 64°C for 30 min, then at 95°C for 5 min to inactivate the enzyme. The reaction product was electrophoresed on a 2% agarose gel. If a ladder-shaped band appeared, it was indicated that there was a LAMP reaction, and if there was no ladder-shaped band, it was indicated as no reaction.

[0037] Detection limit test results: HP strains have...

Embodiment 2

[0039] Cytotoxin-associated protein in Helicobacter pylori ( cagA ) Reaction temperature optimization

[0040] 1 ng of HP strain ATCC 700392 DNA was used as template.

[0041] LAMP reaction system includes: 1×ThermoPol reaction buffer; 6 mM MgSO4; 0.8 M betaine; 1.4 mMdNTPs; the above three pairs of primers, 0.2 μM FIP / BIP; 1.6 μM F3 / B3; 0.8 μM LF / LB; Bst DNA polymerase 320U / mL; DNA template 1 μL, make up to 20 μL with enzyme-free water. For the blank control group, no DNA was added, and 1 μL of enzyme-free water was added accordingly.

[0042] React in water baths at 60°C, 62°C, 64°C, 66°C, 68°C, and 70°C for 30 minutes respectively, and then react at 95°C for 5 minutes to inactivate the enzyme. The reaction product was electrophoresed on a 2% agarose gel. If a ladder-shaped band appeared, it was indicated that there was a LAMP reaction, and if there was no ladder-shaped band, it was indicated as no reaction.

[0043]Reaction temperature optimization results: HP strain ...

Embodiment 3

[0045] Optimization of reaction time of cytotoxin-associated protein (cagA) in Helicobacter pylori

[0046] 1 ng of HP strain ATCC 700392 DNA was used as a template.

[0047] LAMP reaction system includes: 1× ThermoPol reaction buffer; 6 mM MgSO 4 ; 0.8 M betaine; 1.4 mMdNTPs; the above three pairs of primers, 0.2 μM F3 / B3; 1.6 μM FIP / BIP; 0.8 μM LF / LB; Bst DNA polymerase 320U / mL; DNA template 1 μL, make up to 20 μL with enzyme-free water. For the blank control group, no DNA was added, and 1 μL of enzyme-free water was added accordingly.

[0048] React in a water bath at 63°C for 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, respectively, and then react at 95°C for 5 min to inactivate the enzyme. The reaction product was electrophoresed on a 2% agarose gel. If a ladder-shaped band appeared, it was indicated that there was a LAMP reaction, and if there was no ladder-shaped band, it was indicated as no reaction.

[0049] Reaction time optimization results: the produc...

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Abstract

The invention discloses an LAMP primer group for detecting helicobacter pylori cytotoxin related protein cagA and application thereof. The LAMP primer group comprises an inner primer FIP / BIP, an outerprimer F3 / B3 and a loop primer LF / LB. The primer group disclosed by the invention has the technical effects and advantages that the operation is simple: only one water bath kettle is needed to complete the reaction, and no complicated instruments and professional operators are needed; the reaction is fast and efficient: the reaction only needs 30 min; the specificity is high: three pairs of primers are set for the cagA gene, whole gene sequences are fully covered, the triple guarantee enables the pertinence to be very high, and through the DNA verification of other eleven common intestinal strains, no false positive problem exists; and the sensitivity is high: the minimum detection limit is 100 fg DNA.

Description

technical field [0001] The invention belongs to the technical field of biology, and more specifically relates to a method for detecting helicobacter pylori cytotoxin-associated protein cagA The LAMP primer set and its application. Background technique [0002] Long-term infection with HP will increase the incidence of stomach diseases, especially chronic gastritis, duodenal ulcer, gastric cancer, etc. In 1994, the World Health Organization / International Agency for Research on Cancer (WHO / IARC) listed it as I Carcinogens. But not all patients with HP will develop the disease. Studies have shown that genes containing cytotoxin-associated protein A ( cagA ) HP strains are more pathogenic. Cytotoxin-associated protein A (CagA) is produced by cagA Gene encoding, is the main virulence factor of HP. After CagA enters the gastric epithelial cells, it can react with various proteins in the cells, causing gastric cell dysfunction, and even pathological changes in gastric epithel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2565/125
Inventor 肖传兴杨璐溪李晓燕
Owner 厦门蓝特生物科技有限公司
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