CN108611407A - A kind of helicobacter pylori high throughput bacterium colony PCR rapid analysis methods - Google Patents

Abstract

The invention provides a high-throughput colony PCR rapid analysis method for Helicobacter pylori. The invention relates to the field of molecular biology of genes, in particular to the field of PCR amplification of Helicobacter pylori genes. The optimized Helicobacter pylori gene PCR amplification method can realize direct PCR amplification of Helicobacter pylori cells or colonies in a fast and high-throughput manner, and does not require a large number of cultured bacteria to extract its genome. This method improves the efficiency of Helicobacter pylori gene amplification. It is especially suitable for the detection and analysis of the genotype of a large number of samples of Helicobacter pylori.

Description

A high-throughput colony PCR rapid analysis method for Helicobacter pylori

technical field

The invention relates to the field of gene molecular biology, in particular to a high-throughput colony PCR rapid analysis method for Helicobacter pylori.

Background technique

Helicobacter pylori is the main pathogen causing human chronic gastritis, gastric ulcer, gastric mucosal tissue lymphoma and gastric cancer. The infection rate of Helicobacter pylori in the population is very high, more than 50% of the world's population is infected, and the number of infected people in China is as high as about 60%. Helicobacter pylori infection has the characteristics of globalization, infectivity and carcinogenicity, and seriously threatens human health. The World Health Organization has listed it as a class I carcinogen. Significance.

To identify the genotype of Helicobacter pylori by PCR amplification method in the laboratory, or to screen target mutants from a large number of transformants, it is usually necessary to extract its total genome as a template for PCR amplification. A large number of enrichment cultures are carried out for a sample or each transformant, and the total genome is extracted after the bacteria are collected, and then PCR amplification and analysis are performed. This method is time-consuming, laborious, and costly, and it is difficult to apply to multiple samples. Parallel PCR amplification.

Most Gram-negative bacteria, such as Escherichia coli, can directly use their cells or colonies as templates for PCR amplification, without the need for mass culture and collection of cells to extract genomes. However, it is difficult to obtain positive results when directly using Helicobacter pylori cells or colonies for PCR amplification. Conventional cell or colony PCR methods are not suitable for the genome amplification of Helicobacter pylori, which is not conducive to the rapid identification of Helicobacter pylori genotypes. , High-throughput identification and analysis.

Contents of the invention

The object of the present invention is to overcome above-mentioned shortcoming and provide a kind of fast, high-throughput detection and analysis Helicobacter pylori gene, easy to operate Helicobacter pylori high-throughput colony PCR rapid analysis method.

A high-throughput colony PCR rapid analysis method for Helicobacter pylori of the present invention comprises the following steps:

(1) Add 10 μL of deionized water or saline to a 0.2ml PCR tube or a 96-well PCR plate, pick up the cells or colonies of Helicobacter pylori with a toothpick, dissolve and mix well;

(2) Put the well-mixed PCR tube or 96-well PCR plate containing Helicobacter pylori directly into boiling water and heat. When the Helicobacter pylori cells or colonies are boiled and lysed, the cell concentration is less than 1×10 ⁹ CFU/mL, The time for boiling and cracking is not less than 10s. After boiling and cracking, quickly place it on ice and cool it for later use;

(3) Add 1.25 μL each of the forward primer and the reverse primer at a concentration of 10 μM to the above-mentioned lysed cell suspension, add 12.5 μL of 2× premixed Taq DNA polymerase, and the total reaction system is 25 μL. Mix well and place in a PCR machine for polymerase chain reaction. The PCR reaction program is: pre-denaturation at 94°C for 5 minutes, followed by the following cycles: denaturation at 94°C for 30 s; annealing at 60°C for 30 s; extension at 72°C for 1 min; 30 cycles, Final extension at 72°C for 10 min. After PCR, 5 μL of the amplified product was taken for agarose gel electrophoresis detection.

In the high-throughput colony PCR rapid analysis method for Helicobacter pylori above, wherein: the Helicobacter pylori cells or colonies refer to cells derived from in vitro culture or colonies derived from solid medium.

Compared with the prior art, the present invention has obvious beneficial effects. From the above technical solutions, it can be seen that the present invention lyses the thalline by the boiling method, uses the Helicobacter pylori thalline lysate as a template, does not need to extract the genome, and can directly carry out colony extraction. PCR amplification, this method can directly perform colony PCR amplification in a 96-well plate, and can realize parallel PCR detection of multiple samples at the same time, and the PCR amplification products can be recycled for downstream detection and analysis. The invention has the advantages of fast speed and high throughput, provides a new method for the screening, identification and genetic engineering research of Helicobacter pylori, and is especially suitable for screening target bacterial strains from a large number of transformants.

Description of drawings

Figure 1. Agarose gel electrophoresis of colony PCR amplified Helicobacter pylori cagA upstream gene fragment

Figure 2. Agarose gel electrophoresis of colony PCR amplified Helicobacter pylori cagA downstream gene fragment

Figure 3. Agarose gel electrophoresis of colony PCR amplification of Helicobacter pylori 16SrDNA gene

Figure 4. Agarose gel electrophoresis of colony PCR amplification of Helicobacter pylori ureA gene

Figure 5. Agarose gel electrophoresis of colony PCR amplification of Helicobacter pylori iceA gene

Figure 6. Agarose gel electrophoresis of colony PCR amplification of Helicobacter pylori Hp0792 gene

Figure 7. Agarose gel electrophoresis of colony PCR amplification of Helicobacter pylori hetA gene

Figure 8. Agarose gel electrophoresis of colony PCR amplification of Helicobacter pylori ropN gene

Detailed ways

Examples 1-8

A high-throughput colony PCR rapid analysis method for Helicobacter pylori comprises the following steps:

(1) Add 10 μL of deionized water or saline to a 0.2ml PCR tube or a 96-well PCR plate, pick up the cells or colonies of Helicobacter pylori with a toothpick, dissolve and mix well;

(2) Put the well-mixed PCR tube or 96-well PCR plate containing Helicobacter pylori directly into boiling water and heat. When the Helicobacter pylori cells or colonies are boiled and lysed, the cell concentration is less than 1×10 ⁹ CFU/mL, The time for boiling and cracking is not less than 10s. After boiling and cracking, quickly place it on ice and cool it for later use;

(3) Add 1.25 μL each of the forward primer and the reverse primer at a concentration of 10 μM to the above-mentioned lysed cell suspension, add 12.5 μL of 2×Taq premixed DNA polymerase, and the total reaction system is 25 μL. Mix well and place in a PCR machine for polymerase chain reaction. The PCR reaction program is: pre-denaturation at 94°C for 5 minutes, followed by the following cycles: denaturation at 94°C for 30 s; annealing at 60°C for 30 s; extension at 72°C for 1 min; 30 cycles, Final extension at 72°C for 10 min. After PCR, 5 μL of the amplified product was taken for agarose gel electrophoresis detection.

Utilize the scheme of the present invention to respectively amplify 8 randomly selected Helicobacter pylori genes (embodiments 1-8), these 8 genes are respectively: cagA upstream fragment, cagA downstream fragment, 16S rDNA, ureA, iceA, Hp0792, hetA and ropN gene. PCR amplification result shows (Fig. 1-8), utilizes the scheme of the present invention to obtain target gene band (swimming lane 3-6) quickly and conveniently, is similar to the target band concentration (swimming lane 2) that template obtains with genome, and with When the Helicobacter pylori colony that has not been boiled and lysed was used as a template, the band of the target gene could not be obtained (lane 1). It shows that the scheme of the present invention achieves the purpose of obtaining the target gene, and is convenient, fast and efficient.

Table 1. Primers used in the present embodiment 1-8

Claims (2)

1. a kind of helicobacter pylori high throughput bacterium colony PCR rapid analysis methods, include the following steps：

（1）10 μ L deionized waters or physiological saline is added in 0.2 ml PCR pipes or 96 hole PCR plates, with toothpick picking pylorus Helicobacter thalline or bacterium colony dissolve mixing；

（2）The good PCR pipe containing helicobacter pylori of mixing or 96 hole PCR plates are directly placed into boiling water domain and are heated, pylorus spiral shell Cell concentration when bacillus thalline or bacterium colony boiling lysis is less than 1 × 10⁹ The time of CFU/mL, boiling lysis are not less than 10s, boil It is spare that it is positioned over cooled on ice rapidly after boiling cracking；

（3）In the thalline suspension of above-mentioned cracking, forward primer and the reverse primer for being directly added into a concentration of 10 μ Μ are each 1.25 μ L are added 2 × premix Taq archaeal dna polymerases, 12.5 μ L, react 25 μ L of total system,

It mixes well to be placed in PCR instrument and carries out PCR, 5 μ L amplified productions is taken to carry out agarose after PCR Detected through gel electrophoresis.

2. helicobacter pylori high throughput bacterium colony PCR rapid analysis methods described in accordance with the claim 1, wherein：The pylorus spiral shell Bacillus thalline or bacterium colony refer to the thalline from vitro culture or the bacterium colony on solid medium.