阅读说明：本技术 一种潜伏期胃癌诊断试剂盒 (Latent gastric cancer diagnostic kit ) 是由 廖兴华 黄凤 项园 刘美君 王根鑫 段圆圆 李佳蓬 李慧 张同存 于 2019-11-05 设计创作，主要内容包括：本发明提出了一种潜伏期胃癌诊断试剂盒,包括检测人细胞毒素相关蛋白A表达水平的试剂和能够检测出微生物的试剂,本发明在检测细胞毒素相关蛋白A阳性基础上,抽提粪便中细菌基因组DNA,通过DGGE分析肠道微生物群落多样性,可以判断患胃癌的风险,为患者采取尽早诊断和干预提供有效的依据。(The invention provides a latent gastric cancer diagnostic kit, which comprises a reagent for detecting the expression level of human cytotoxin-related protein A and a reagent capable of detecting microorganisms.)

1. A latent gastric cancer diagnostic kit characterized in that: comprises an agent for detecting the expression level of human cytotoxin-associated protein A and an agent capable of detecting microorganisms including one or more of the genera Fusobacterium (Fusobacterium Nuclear), Lachnospira (Lachnospira Bryant and Small), Veillonella (Veillonella), Clostridium perfringens (Clostridium perfringens), Helicobacter pylori (Helicobacter pylori), Escherichia coli (Escherichia), Streptococcus (Streptococcus), Bifidobacterium (Bifidobacterium), Lactobacillus (Lactobacillus), and Streptococcus faecalis (Streptococcus faecalis).

2. The latent gastric cancer diagnostic kit according to claim 1, wherein: the reagent for detecting the expression level of the human cytotoxin-associated protein A is a reagent for an ELISA detection method.

3. The latent gastric cancer diagnostic kit according to claim 2, wherein: the reagent for detecting the expression level of the human cytotoxin-related protein A comprises an enzyme conjugate, a washing solution, a sample diluent, a color developing agent and a confining solution.

4. The latent gastric cancer diagnostic kit according to claim 1, wherein: the reagent capable of detecting microorganisms includes a reagent capable of extracting bacterial genomic DNA from feces of a subject.

5. The latent gastric cancer diagnostic kit according to claim 4, wherein: the reagent capable of detecting the microorganism comprises lysis solution, eluent, protease, primers, DNA polymerase and dNTPs.

6. Use of the latent gastric cancer diagnostic kit according to claim 1 for screening or identifying a drug for treating gastric cancer.

7. A method of detecting a microorganism, comprising: the method is used to provide information required to predict or diagnose latent gastric cancer, and the microorganisms include one or more of the genera Fusobacterium (Fusobacterium tuberculosis), Lachnospira (Lachnospira Bryant and Small), Veillonella (Veillonella), Clostridium perfringens (Clostridium perfringens), Helicobacter pylori (Helicobacter pylori), Escherichia (Escherichia), Streptococcus (Streptococcus), Bifidobacterium (Bifidobacterium), Lactobacillus (Lactobacillus), and Streptococcus faecalis (Streptococcus faecalis).

8. The method for detecting microorganisms according to claim 7, wherein: comprises the step of extracting bacterial genome DNA in the feces of a tested person.

Technical Field

The invention relates to the technical field of biological medicines, in particular to a latent gastric cancer diagnostic kit.

Background

Gastric cancer is an important cancer worldwide, with over 1,000,000 new cases in 2018, of which about 783,000 deaths (corresponding to 1 per 12 deaths worldwide) make it the fifth most common cancer and the third most deaths. Wherein the morbidity and mortality of men are 1.5-2 times higher than those of women. Since patients with early-stage gastric cancer often have no symptoms or only slight symptoms, and when clinical symptoms are obvious, lesions belong to middle and late stages, the early-stage symptoms of gastric cancer need to be very vigilant to avoid delaying diagnosis and treatment.

The common method for examining stomach diseases is gastroscopy, and if abnormality is found, pathological examination of tissue sections is performed. Gastroscopy causes certain pain to patients, requires precise operation and strict disinfection, otherwise causes damage and cross infection to the patients, and has high cost, so most latent gastric cancer patients do not select to carry out the gastroscopy for a plurality of times regularly when no obvious symptoms exist, and treatment delay and life danger can be caused.

The pathogenesis of gastric cancer is not known at present, but the harmful environment, the intake of preserved food and the intake of low fruit increase the risk of gastric cancer, and drinking and smoking are established risk factors, wherein the infection of Helicobacter pylori (Helicobacter pylori) is the main risk factor for gastric cancer, nearly 90% of cases of non-cardiac gastric cancer are attributed to the bacterium, and the world health organization has defined Helicobacter pylori as a class I carcinogen. The balance of intestinal microbial flora is influenced by factors such as diet, medicines, environment and the like, and plays an important role in stabilizing the environment in the intestinal tract. Beneficial bacteria and pathogenic bacteria in the intestinal tract of a human body compete with each other to maintain the balance in the intestinal tract, and when the pathogenic bacteria are increased greatly, the balance of the intestinal mucosa immune system is broken, so that intestinal inflammation is possibly caused. The imbalance of the intestinal flora is considered to be an important factor influencing the occurrence and development of various diseases, so that the diseases can be prevented, the health is promoted, the symptoms are improved, and the disease course is shortened by regulating the intestinal flora.

Through the research on the correlation of several specific genes of helicobacter pylori, the cytotoxin-related gene A (cagA) is considered to be a main virulence gene causing cell canceration, and after the cytotoxin-related protein A coded by the cagA gene enters gastric epithelial cells, the cytotoxin-related protein A can react with a plurality of proteins in the cells to cause the functional disorder of the cells and even cause the pathological change of the gastric epithelial cells, thereby causing diseases. Various studies have shown that infection with CagA-positive H.pylori increases the risk of atrophic gastritis and gastric cancer.

Disclosure of Invention

In view of the above, the invention provides a latent gastric cancer diagnostic kit, which can detect the expression level of human cytotoxin-related protein A and the diversity of intestinal microflora, so that the risk of gastric cancer can be judged more accurately.

The technical scheme of the invention is realized as follows:

in a first aspect, the present invention provides a latent gastric cancer diagnostic kit comprising an agent for detecting the expression level of human cytotoxin-associated protein a and an agent capable of detecting microorganisms including one or more of Fusobacterium (Fusobacterium tuberculosis), Lachnospira (Lachnospira Bryant and Small), Veillonella (Veillonella), Clostridium perfringens (Clostridium perfringens), helicobacter pylori (helicobacter), Escherichia (Escherichia), Streptococcus (Streptococcus), Bifidobacterium (Bifidobacterium), Lactobacillus (Lactobacillus), and Streptococcus faecalis (Streptococcus faecalis).

On the basis of the above technical solution, preferably, the reagent for detecting the expression level of human cytotoxin-associated protein a is a reagent for an ELISA detection method.

Further, preferably, the reagent for detecting the expression level of the human cytotoxin-associated protein A comprises an enzyme conjugate, a washing solution, a sample diluent, a color developing agent and a sealing solution.

In addition to the above technical means, preferably, the reagent capable of detecting microorganisms includes a reagent capable of extracting bacterial genomic DNA from feces of a subject.

Further, preferably, the reagent capable of detecting microorganisms includes a lysis solution, an elution solution, a protease, a primer, a DNA polymerase and dNTPs.

In a second aspect, the invention provides an application of the latent gastric cancer diagnostic kit in screening or identifying drugs for treating gastric cancer.

In a third aspect, the present invention also provides a method of detecting microorganisms including one or more of the genera Fusobacterium (Fusobacterium tuberculosis), Lachnospira (Lachnospira Bryant and Small), Veillonella (Veillonella), Clostridium perfringens (Clostridium perfringens), Helicobacter pylori (Helicobacter pylori), Escherichia (Escherichia), Streptococcus (Streptococcus), Bifidobacterium (Bifidobacterium), Lactobacillus (Lactobacillus), and Streptococcus faecalis (Streptococcus faecalis) for providing information required to predict or diagnose latent gastric cancer.

On the basis of the above technical solution, preferably, the method for detecting microorganisms includes the step of extracting bacterial genomic DNA from feces of a subject.

Compared with the prior art, the latent gastric cancer diagnostic kit has the following beneficial effects:

(1) the kit can detect the expression level of the CagA protein of the helicobacter pylori and the composition of intestinal microbial flora, and can more accurately predict early gastric cancer by detecting whether the intestinal microbial community is dysregulated or not on the basis of judging the CagA to be positive;

(2) the method for identifying the microorganisms commonly used in hospitals adopts a traditional culture method, is long in time consumption, high in culture requirement and multiple in influencing factor, more importantly, is difficult to simulate the conditions of natural environment, so that some bacteria cannot obtain pure cultures, in addition, the culture method only can culture live bacteria, but dead bacteria cannot count, however, the method for identifying the microbial flora by detecting the bacterial genome DNA in the excrement has the advantages of multiple obtained strains, more accurate result, convenience, rapidness and high sensitivity.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Subject information was registered and 25 male and female patients with intestinal metaplasia and dysplasia were selected as a patient group, 50 of which were diagnosed with gastric cancer at a later time, 25 healthy male and female patients were selected as a control group, and the ages of both groups were between 42 and 48 years.

S1 detection of protein A (CagA) associated with cytotoxin

Urine samples of testees of a patient group and a control group are collected, and the content of CagA protein in the urine is detected by using a human cytotoxin associated protein A (CagA) ELISA kit of Wuhan Moishak.

Sample preparation: 10mL of urine is collected by a sterile tube, centrifuged at 3000r/min at 4 ℃ for 20min, and the supernatant is carefully collected, and centrifuged again if a precipitate is generated during the storage.

Dilution of concentrated sample diluent: the concentrated sample dilution was diluted 10-fold with distilled water for use.

Sample adding: and respectively arranging a blank hole, a standard hole and a sample hole to be detected on the enzyme-labeled coated plate, respectively and accurately adding 50 mu L of diluted sample diluent and 50 mu L of standard substance into the blank hole and the standard hole, and adding 40 mu L of diluted sample diluent and then 10 mu L of sample to be detected into the sample hole to be detected. The sample is not contacted with the hole wall as much as possible during sample adding, and the sample is gently shaken to be uniform after sample adding.

And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 min.

Preparing liquid: the 30 times of concentrated washing liquid is diluted by 30 times of distilled water for standby.

Washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling each hole with a cleaning solution, standing for 30s, discarding, and patting dry.

Adding an enzyme: 50 μ L of enzyme conjugate was added to each well, except for blank wells.

And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 min.

Washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling each hole with a cleaning solution, standing for 30s, discarding, and patting dry.

Color development: adding 50 μ L of color-developing agent A into each well, adding 50uL of color-developing agent B, shaking gently, mixing, and developing at 37 deg.C in dark for 15 min.

And (4) terminating: the reaction was stopped by adding 50. mu.L of blocking solution to each well, whereupon the color in the wells immediately turned yellow.

And (3) determination: the blank wells were zeroed, and the absorbance (OD) of each well was measured at a wavelength of 450nm, and the measurement should be performed within 15min after the addition of the stop solution.

The experimental results are as follows:

when the CagA is more than 150mg/L, the judgment is positive, the CagA content of the patient with the intestinal metaplasia and dysplasia is obviously higher than that of a healthy control group, the significant difference is achieved (P is less than 0.05), and the possibility of suffering from gastric cancer is obviously improved due to the high expression of the cytotoxin-related protein A.

S2, detecting intestinal microorganisms

1) 200mg of excrement samples of patients and control group testers are respectively weighed and put into a sterile test tube to be placed on ice, bacterial genome DNA of all samples is extracted by using a DNA extraction kit of the Foregene company, and the extracted DNA samples are frozen in a refrigerator at the temperature of-20 ℃.

2) PCR amplification

The 16S rRNA gene V3 variable region sequence of the flora genome is amplified, and the sequence of the universal primer is as follows: an upstream primer: GC-341F (5 '-GC clamp-CCTACGGGAGGCAGCAG-3'), downstream primer: 518R (5'-ATTACCGCGGCTGG-3') with a "GC clamp" of 40bp and a sequence of CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGG.

PCR amplification was carried out using the PCR kit of Shanghai assist in san Francisco. PCR amplification System (50. mu.L): 8 μ L10 XPCR mix Buffer (Mg) ²⁺) 5 μ L of 1% BSA, 5 μ L of dNTPs, 1 μ L of each of the upstream and downstream primers, 3 μ L of template DNA, 0.5 μ L of Taq DNA polymerase (5U/. mu.L), and 26.5 μ L of deionized water. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 6 min; denaturation at 95 ℃ for 30 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 30 s; 30 cycles; fully extending for 10min at 72 ℃. After the reaction is finished, the product is stored on ice or at 4 ℃. After the PCR was completed, the PCR product was subjected to 1% agarose gel electrophoresis using 5. mu.L of the PCR product to examine whether the PCR was successful or not.

3) Denaturing Gradient Gel Electrophoresis (DGGE)

After 20. mu.L of the PCR product was mixed with 5. mu.L of 6 × Loading Buffer, DGGE electrophoresis analysis was performed using a denaturing gradient gel electrophoresis system. According to the preliminary experiment, the concentration gradient of the denatured gel is set to be 30% -55%, electrophoresis is carried out in a thermostatic water bath at 60 ℃, the high pressure impact is carried out at 140V for 20min, then the low pressure is changed to 60V, and the used electrophoresis Buffer is 1 × TAE Buffer. After electrophoresis, the gel is stained for 10min on a shaking table by using EB staining solution and detected in a gel imaging system.

4) DGGE profiling

Cutting the differential bands into sterile EP tubes, washing 3 times with sterile water, blotting the supernatant, and adding 50 μ L of 3dH ₂O-20 ℃ refrigerator overnight storage. The next day, water bath at 90 ℃ for 10min, centrifugation at 10000 Xg for 5min, taking 3 μ L of supernatant as a template, 341F without GC clamp as an upstream primer, and carrying out PCR amplification on the rest components and conditions in the same step 2). The amplified PCR product was subjected to 1% agarose electrophoresis, and the amplified DNA band was recovered by cutting the gel according to the instructions of a general agarose gel DNA recovery kit of TIANGEN. The purified DNA samples were sent to TaKaRa (Wuhan) for sequencing and the sequencing results were compared in NCBI database. And (3) applying Quantity One software to perform strip number, similarity analysis and clustering analysis on the DGGE map.

The experimental results are as follows:

the 16S rDNA V3 region PCR amplification products of the patient group and the control group are detected by 1% agarose gel electrophoresis, and the results show that the PCR products are both 200bp and have no non-specific amplification fragments.

And carrying out DGGE electrophoresis on the PCR products, analyzing the difference bands, and identifying the composition of the intestinal microflora of the patient group and the control group. The differences in the dominant intestinal microbial flora between the patient groups and the control groups by sequencing and comparison with the NCBI database are shown in the following table.

As shown by the above table, the dysbacteriosis is severe in patients with intestinal metaplasia and dysplasia, with male patients more severe than female patients. Therefore, the risk of gastric cancer can be judged by detecting the diversity of intestinal microflora.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.