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Primer, probe, method and kit for detecting helicobacter suis

A technology of Helicobacter suis and Helicobacter, which is applied in the field of molecular biology, can solve the problems of slow progress and difficulties in the isolation and cultivation of Helicobacter suis, and achieve the effect of good accuracy and fast detection speed

Pending Publication Date: 2022-06-07
苏州长柏生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the difficulty in the isolation and cultivation of Helicobacter suis, its molecular biological detection is of great significance, but this work has been progressing slowly

Method used

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  • Primer, probe, method and kit for detecting helicobacter suis
  • Primer, probe, method and kit for detecting helicobacter suis
  • Primer, probe, method and kit for detecting helicobacter suis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Optimization of annealing temperature of Helicobacter pig real-time PCR system: the system annealing temperature changed from 55°C to 62°C, and the results showed that the amplification effect of the system was the best at about 58°C ( Figure 1 )

[0050] Optimization of HS real-time PCR amplification system and amplification conditions

[0051] System configuration table

[0052]

[0053]Amplification conditions:

[0054] 95°C 10min, 45 cycles: 95°C 10s, 55°C~62°C 30s.

Embodiment 2

[0056] The production of system standard curves

[0057] The real-time PCR standard curve of hp alpB system is plotted with the log value of the standard concentration template as the abscissa and the corresponding Ct value as the ordinate. The results show that when the positive template amount is in the range of 17.8fg~17.8ng, its logarithmic value has a very good correlation with the Ct value (R2=0.997). see Figure 2 。

Embodiment 3

[0059] Evaluation of sensitivity, specificity and detection limit of HS fluorescent PCR detection system

[0060] 1. Clinical isolate test verification

[0061] Because Helicobacter pig is currently isolated very little in the world, we actually tested a clinically isolated strain of DNA with an optimized HS detection system for Helicobacter suis. As a result, there was no amplification except NTC, and the results of Helicobacter suis were positive.

[0062] 2. Specificity evaluation

[0063] Specificity, also known as true negative rate, is the percentage of Helicobacter pig that is not actually Helicobacter pig but is correctly judged according to the standards of this detection method. 20 common strains of the digestive tract and human chromosomes were detected with optimized real-time PCR (Table 1), with Helicobacter pig as the positive control. In addition to the positive control, the remaining 21 non-Helicobacter pigs templates were negative. see Figure 3 。

[0064] Table 1 ...

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Abstract

Aiming at specific gene analysis of the porcine helicobacter, the invention provides a real-time fluorescent quantitative PCR primer and a probe for detecting the porcine helicobacter, and nucleotide sequences of the primer and the probe are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The invention further provides a method and a detection kit for quantitatively detecting the helicobacter suis. The detection method disclosed by the invention has the advantages of accurate detection, high sensitivity, strong specificity, simplicity, convenience and rapidness in operation and good actual specimen detection capability.

Description

Technical field [0001] The present invention relates to the field of molecular biology, in particular to primers and probes for detecting Helicobacter holi by fluorescence quantitative PCR, the present invention further relates to methods and kits for detecting Helicobacter boris by using the primers and probes. Background [0002] Helicobacter suis is a zoonotic pathogen that infects not only pigs but also humans. On a global scale, Helicobacterium viviensis is more than 60% of pig infections, and infections are found in pigs of all ages, mainly causing ulcers in the stomach (esophagus area) of pigs. As we all know, Helicobacter pylori is the main pathogenic agent of human stomach diseases and is the class I carcinogenic factor specified by the Cancer Agency of the World Health Organization. Related research surface, in addition to Helicobacter holium colonization in the human stomach, there are other Helicobacter colonization (Non-Helicbacter pylori helicobacters, NHPH), the mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6851C12Q2600/166C12Q2600/158C12Q2531/113C12Q2563/107C12Q2545/113Y02A50/30
Inventor 刘国栋宋衍燕聂艳妮
Owner 苏州长柏生物科技有限公司