Kit for rapidly detecting helicobacter pylori and application

Abstract

The invention discloses a kit for rapidly detecting helicobacter pylori and application. A forward primer of the helicobacter pylori is as shown in SEQ ID NO. 1; a reverse primer of the helicobacter pylori is as shown in SEQ ID NO.2, and a probe sequence of the helicobacter pylori is as shown in SEQ ID NO.3. The specific RPA amplification primer group is designed and developed, and an amplicon with a mark can be combined with a modifier on a lateral flow chromatography test strip, so that the helicobacter pylori detection is facilitated, and the specificity and the sensitivity are relatively high. According to the invention, the detection sensitivity can reach 50 copies / T. In the detection process, the kit provided by the invention is simple in material taking, low in requirements on test environment and operating skills of personnel, simple and convenient to operate, capable of being skillfully operated through simple training, low in requirements on detection equipment, convenient to popularize and use in basic levels in outdated remote areas, and wide in application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology detection, and specifically relates to a rapid detection kit for Helicobacter pylori and its application, and especially to a visual detection technology for Helicobacter pylori nucleic acid. Background Art

[0002] Helicobacter pylori, scientifically known as Helicobacter pylori, is a Gram-negative, spiral-shaped bacterium that is one of the main causes of stomach diseases such as gastric ulcers and gastric cancer. Infection with Helicobacter pylori is usually transmitted through oral-fecal or oral-oral transmission, and may cause gastritis, peptic ulcers and other diseases after infection.

[0003] The detection techniques of Helicobacter pylori include traditional methods such as breath testing, gastroscopy, tissue biopsy, etc., as well as molecular biological methods such as PCR, in situ hybridization, etc. However, traditional detection methods usually require professional personnel to operate, are time-consuming and labor-intensive, and have high costs. Some methods require invasive sampling, such as gastroscopy or tissue biopsy, which is not very patient-friendly and has certain risks. Some methods have limitations in sensitivity and specificity, which may lead to false negative or false positive results. Some molecular biological methods require complex experimental operations and equipment, which may not be suitable for general medical institutions or remote areas. Therefore, the development of simple, rapid, accurate, low-cost Helicobacter pylori detection technology that is suitable for various environments remains the current research focus. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention introduces recombinase polymerase amplification technology (RPA) in the qualitative detection of Helicobacter pylori, and successfully develops a highly specific and highly sensitive Helicobacter pylori nucleic acid detection kit by designing and developing a specific RPA amplification primer set for Helicobacter pylori Helicobacter pylori gene. This kit is not only easy to obtain materials during the detection process, but also has low requirements for the experimental environment and the skills of the operator, and can be operated proficiently after simple training. In addition, the kit has low requirements for detection equipment, so it can easily realize detection under the condition of lack of equipment, showing a very broad application prospect. Not only improves the detection efficiency and accuracy, but also broadens the detection scene.

[0005] To achieve the above objectives, the present invention first designs a primer probe set for rapid detection of Helicobacter pylori, the primer probe set comprising a Helicobacter pylori forward primer, a Helicobacter pylori reverse primer and a Helicobacter pylori probe;

[0006] The nucleotide sequence of the Helicobacter pylori forward primer is shown in SEQ ID No. 1;

[0007] The nucleotide sequence of the Helicobacter pylori reverse primer is shown in SEQ ID No. 2;

[0008] The nucleotide sequence of the Helicobacter pylori probe is shown in SEQ ID No.3.

[0009] Preferably, the 5' end of the Helicobacter pylori reverse primer is labeled with biotin.

[0010] Preferably, the 5' end of the H. pylori probe is modified with an antigenic molecule marker; the 3' end of the H. pylori probe is modified with a polymerase extension blocking group to prevent amplification extension, which can be any one of phosphate, C3-Spacer, amine or dideoxynucleotide, more preferably C3-Spacer.

[0011] The antigenic molecule marker can be biotin, tetramethylrhodamine, digoxin, FAM, FITC, and FAM is more preferred. In some other specific embodiments, the antigenic molecule marker can also be any other antigenic molecule marker that matches the antibody or specific binding molecule on the lateral flow chromatography test strip.

[0012] In an embodiment of the present invention, the Helicobacter pylori probe is an nfo probe structure, the middle section of which is modified with an internal abasic nucleotide analog (tetrahydrofuran [THF] residue) as a nucleotide substitute; it is located at the 18th base from the 3' end of the middle of the probe nucleotide sequence by replacing the original nucleotide, and the THF residue replaces the nucleotide that can normally perform base pairing with the complementary sequence as a recognition site for the nuclease.

[0013] Another aspect of the present invention provides a kit for rapid detection of Helicobacter pylori, the kit comprising a primer probe set for amplifying a DNA sample to be tested; the primer probe set is the primer probe set or its complementary chain, or has a single sequence homology of 50%, 60%, 70%, 80%, 90%, 95%, 99% or more to the primer probe set or its complementary chain; in some more preferred embodiments, the primer probe set for amplifying a DNA sample to be tested has the following sequence:

[0014] The nucleotide sequence of the forward primer is shown in SEQ ID No. 1;

[0015] The nucleotide sequence of the reverse primer is shown in SEQ ID No. 2;

[0016] The nucleotide sequence of the probe is shown in SEQ ID No.3.

[0017] In some more preferred embodiments, the kit further comprises a nucleic acid extraction reagent, a RPA amplification reaction freeze-dried buffer ball, an RPA freeze-dried enzyme Mix ball, and a lateral flow chromatography test strip;

[0018] The lateral flow chromatography test strip comprises a sample loading pad area, a colloidal gold labeled antibody area, a nitrocellulose membrane layer, and a reaction chromatography membrane absorption area; the nitrocellulose membrane layer is provided with a test line sprayed with streptavidin and a quality control line sprayed with antibodies; the colloidal gold labeled antibody area is provided with colloidal gold labeled anti-FAM antibodies.

[0019] In view of the role of the primer probe set provided by the present invention, the use of the primer probe set in the preparation of products for detecting Helicobacter pylori also falls within the protection scope of the present invention.

[0020] Preferably, the RPA amplification reaction freeze-dried buffer ball is polyethylene glycol 1.5-5wt%; Tris 10-50mM; sodium acetate 200-240mM; dithiothreitol 1-10mM; ATP 1.5-3.5mM; creatine phosphate disodium salt 20-75mM; phosphokinase 10-200ng / μL; deoxyribonucleoside triphosphate, 450-500μM; trehalose 2.5-7.5wt%; mannitol 40-50ng / μL; activator magnesium acetate; the RPA freeze-dried enzyme Mix ball is a recombinase, a single-stranded DNA binding protein, a DNA polymerase and a nuclease.

[0021] Preferably, the amplification temperature is 37-42°C and the amplification time is 15-20 min.

[0022] Another aspect of the present invention further provides a method for using the above-mentioned kit for rapid detection of Helicobacter pylori, comprising the following steps:

[0023] The above primer probe set is used to amplify the DNA of the sample to be tested, and then the sample to be tested is tested using a lateral flow chromatography test strip. If the test line shows a band, the sample to be tested contains Helicobacter pylori, and if no band is shown, the sample to be tested does not contain Helicobacter pylori.

[0024] The method can detect samples including dental plaque, etc.; the sensitivity of the method to the DNA of the sample to be tested is 50 copies / T.

[0025] The more specific steps are:

[0026] Use nucleic acid extraction reagent to quickly release DNA from the sample to be tested, and add the RPA amplification reaction freeze-dried buffer ball, RPA freeze-dried enzyme Mix ball and the sample DNA to be tested in the kit to the RPA reaction system;

[0027] The preferred RPA reaction system is 50 μL, specifically including: RPA amplification reaction freeze-dried buffer ball, RPA freeze-dried enzyme Mix ball, 2 μL each of forward and reverse primers (10 uM), 0.5 μL of nfo probe (10 uM), and 47.5 μL of the sample DNA template to be tested; mix evenly in a reaction tube.

[0028] The RPA amplification product is diluted 20 times with PBST to form an amplification product solution, and a lateral flow chromatography test strip is inserted into the amplification product solution. After 5-10 minutes, the test strip is taken out for observation. The quality control line sprayed with antibody is colored, indicating that the test strip is effective; at the same time, a detection line sprayed with streptavidin is provided on the nitrocellulose membrane layer. If the color line is detected at this place, the sample to be tested contains Helicobacter pylori; if the color line is not detected at this place, the sample to be tested does not contain Helicobacter pylori.

[0029] The principle of the present invention is that the nfo probe specifically binds to the DNA to be tested to form a complex with two labels (biotin and FAM); the complex is then dripped into the sample addition area of ​​the test strip and combined with the colloidal gold particles (with FAM antibodies) in the sample addition area. The newly formed complex diffuses to the detection line under the diffusion action of the chromatographic membrane, and only the complex labeled with biotin will be captured by the biotin ligand, thereby making the detection line "colorful", while the colloidal gold particles not bound to the test object will continue to diffuse to the quality control line and be captured by the specific antibody, thereby making the quality control line "colorful".

[0030] Compared with the prior art, in order to realize a rapid detection of Helicobacter pylori, the present invention provides a primer probe set for Helicobacter pylori based on RPA constant temperature nucleic acid rapid amplification technology, taking the urease Urea gene of Helicobacter pylori as the detection target, designing specific primers in the conserved region of the gene, amplifying the detection target gene fragment by RPA constant temperature rapid amplification technology, and then detecting the amplified product with the help of lateral flow chromatography test strips. The primer probe set designed by the present invention is to select the primer probe set with the highest specificity, which can efficiently and selectively amplify the nucleotide sequence of the conserved region of Helicobacter pylori. The present invention can be applied to oral dental plaque, has the advantages of convenient and fast sampling, greatly reduces the dependence on professional equipment, and can obtain the test results within 20 minutes. The sensitivity of the present invention is high, and the sensitivity of specific detection of Helicobacter pylori can reach 50 copies / μL, which is consistent with the test results of qPCR. DETAILED DESCRIPTION

[0031] The present invention is further described by the following examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention according to the above invention content and implement them specifically, which still falls within the scope of protection of the invention.

[0032] Example 1

[0033] The present embodiment provides a rapid Helicobacter pylori test kit, comprising a nucleic acid extraction reagent, an RPA amplification reaction freeze-dried buffer ball, an RPA freeze-dried enzyme Mix ball, and a lateral flow chromatography test strip.

[0034] RPA amplification reaction freeze-dried buffer balls and RPA freeze-dried enzyme Mix balls were purchased from Anpu Future Biotechnology Co., Ltd. The RPA freeze-dried enzyme Mix balls include recombinase, single-stranded DNA binding protein, DNA polymerase and exonuclease. The exonuclease is used to recognize and cut the THF site in the Helicobacter pylori probe.

[0035] The RPA amplification system includes a Helicobacter pylori forward primer, a Helicobacter pylori reverse primer, a Helicobacter pylori probe, a freeze-dried buffer ball, and an RPA freeze-dried enzyme Mix ball.

[0036] As a reference, RPA amplification reaction freeze-dried buffer ball, RPA freeze-dried enzyme Mix ball, 2 μL each of forward and reverse primers (10 uM), 0.5 μL of nfo probe (10 uM), and 47.5 μL of the sample DNA template to be tested.

[0037] As a reference, the present invention accurately locked the base sequence markers of the specific pathogen target gene through the published Helicobacter pylori gene sequence (GenBank). On this basis, the present invention comprehensively used PrimerPremier 5, DNAMAN software and NCBI's Primer-BLAST platform, and the primer probe design principles were carried out according to the principles published on the official website of TwistDx Inc. Both primers and probes were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0038] As a reference, the 5' end of the probe is connected to an antigen molecule, which is any one of biotin, tetramethylrhodamine, digoxigenin, FAM, FITC or other antigen molecules that match the antibody or specific binding molecule on the lateral flow chromatography test strip.

[0039] As a reference, the Helicobacter pylori probe is an nfo probe structure, and the middle of its sequence is modified with an internal abasic nucleotide analog tetrahydrofuran THF residue as a nucleotide substitute; it is located at the 18th base from the 3' end of the middle of the probe nucleotide sequence, and the tetrahydrofuran THF residue replaces the nucleotide that can normally base pair with the complementary sequence, as the recognition site of the nuclease.

[0040] Specifically, as a reference, in this embodiment, the 5' end of the Helicobacter pylori reverse primer is connected to the antigen molecule biotin, and the 5' end of the Helicobacter pylori probe is connected to carboxyfluorescein (FAM). The 3' end of the Helicobacter pylori probe is connected to the blocking group C3-Spacer, and the middle of the Helicobacter pylori probe is modified with a tetrahydrofuran THF residue of the cleavage recognition site.

[0041] More specifically, the present invention designs multiple different primer probe sets, and performs specificity tests on each primer probe set according to the method of Test Example 1. In this example, only the primer probe set with the highest specificity is listed. The nucleotide sequences of the primers and probes used in the examples of the present invention are shown in Table 1.

[0042] The nucleotide sequence of the Helicobacter pylori forward primer is shown in SEQ ID No. 1;

[0043] The nucleotide sequence of the Helicobacter pylori reverse primer is shown in SEQ ID No. 2;

[0044] The nucleotide sequence of the Helicobacter pylori probe is shown in SEQ ID No.3.

[0045] Table 1

[0046]

[0047] In addition, the present scheme also provides a lateral flow chromatography test strip having the following structure: comprising a sample loading pad area, a colloidal gold labeled antibody area, a nitrocellulose membrane layer, and a reaction chromatography membrane absorption area; the nitrocellulose membrane layer is provided with a detection line sprayed with streptavidin and a quality control line sprayed with antibodies; the nanogold particle binding pad has colloidal gold labeled anti-FAM antibodies.

[0048] This scheme also provides an application of a rapid Helicobacter pylori detection kit, including adding a sample to be tested to an RPA amplification system after a nucleic acid extraction reagent is added to obtain an amplification product, and then dropping the amplification product on a lateral flow chromatography test strip to display the test result.

[0049] Test Example 1: Specificity Test

[0050] DNA was extracted from Helicobacter pylori, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Acinetobacter baumannii, Salmonella and Staphylococcus epidermidis and the concentration was set to 10 5 copies / μL.

[0051] The detection steps are as follows:

[0052] (1) Extraction of cross-strain nucleic acid

[0053] A commercial extraction kit (Kangwei Century) was used to extract DNA from cross-bacterial samples.

[0054] (2) Add freeze-dried buffer balls, RPA freeze-dried enzyme Mix balls, 2 μL each of forward and reverse primers (10 μM), and 0.5 μL of nfo probe (10 μM) to the RPA amplification reaction tube. After fully dissolving, add 47.5 μL of the DNA template of the sample to be tested and react at 39°C for 20 min.

[0055] (3) Lateral flow chromatography test strips

[0056] Take 10 μL of amplification product and mix it with 190 μL of distilled water, then immerse the test strip in the mixture, observe the control line and the detection line, and take out the test strip 2 minutes after the liquid reaches the top of the test strip.

[0057] The experimental results showed that the kit of this scheme only detected positive results for Helicobacter pylori, and no abnormal band color was detected for other bacterial species.

[0058] Test Example 2: Sensitivity Test

[0059] The positive control (plasmid containing the H. pylori gene fragment) was diluted to 10 5 , 10 4 , 10 3 , 10 2 , 50, 25 copies / T, and amplification was performed according to the steps of Test Example 1 to detect sensitivity. The results showed that for plasmid 10 containing the Helicobacter pylori gene fragment 5 -10 2 The samples with concentrations of 100 copies / T and 50 copies / T were amplified by the kit of this solution and the color was obvious on the detection line of the test strip. The samples with a concentration of 25 copies / T were not amplified. Therefore, the detection sensitivity of the detection method provided by the present invention was 50 copies / T.

[0060] Test Example 3: Test kit for testing dental plaque samples

[0061] As an optional method, the above-mentioned Helicobacter pylori nucleic acid detection reagent cartridge also includes a dental plaque nucleic acid extract.

[0062] As an optional method, the above-mentioned Helicobacter pylori nucleic acid detection reagent card box also includes a dental plaque collection and storage tube.

[0063] Furthermore, in the dental plaque collection and storage tube:

[0064] (1) a lysis buffer comprising Tween20 or Triton x100 at a concentration of about 0.1% to about 25%, preferably about 2% to about 5%;

[0065] (2) A Tris-HCl buffer having a concentration ranging from about 0.1 M to about 2.0 M.

[0066] As an optional method, in the above Helicobacter pylori nucleic acid detection reagent cartridge, in the saliva collection and storage tube:

[0067] (1) a lysis buffer comprising Tween 20 and Triton x 100 at a concentration of about 0.1% to about 5%;

[0068] (2) Contains about 0.1 M to about 5.0 M Tris hydrochloric acid buffer.

[0069] In order to ensure the accuracy and improve the sensitivity of the test, the quality of the DNA used for the test is very important. Sampling requirements: Do not brush your teeth at night, and take samples before eating the next morning;

[0070] Collect samples in accordance with relevant standards. It is recommended to test samples as soon as possible after collection. Samples tested within 8 hours can be temporarily stored at 4°C. If sample preservation fluid or rapid release agents are used to treat samples, pay attention to the effects of residues of substances that affect protein activity, such as acids, alkalis, salts, and surfactants, on reagent performance.

[0071] Sampling method: Select the four first molars of the six test subjects as sampling teeth. If they are missing, take the second molars. Use a sterile toothpick to scrape the dental plaque from the buccal mesial site of the four molars. Pay attention to standard operation to avoid contamination. Place the swab in the sample processing tube, rotate and squeeze the sampling head to fully mix the sample with the processing liquid.

[0072] The analysis method flow is as follows:

[0073] S1. Release of nucleic acids in positive dental plaque samples to be tested;

[0074] S2, using the processed sample to be tested as a template, amplifying the template using an RPA isothermal amplification method to obtain an RPA amplification product;

[0075] S3. Use a double-labeled colloidal gold immunochromatographic test strip to detect the RPA amplification product, and judge the positive and negative and identify the specific type based on the presence or absence of bands on the test strip.

[0076] Specifically, in step S1, the release or extraction of nucleic acid in the sample to be tested adopts a one-step nucleic acid extraction reagent to release the nucleic acid to form a template to be amplified.

[0077] The specific steps of preparing the template in step S2 include: in the embodiment of the present invention, the sample used is a positive Helicobacter pylori dental plaque sample, and the one-step nucleic acid extraction reagent independently developed by the company is used to release nucleic acid. This sample is the template to be amplified.

[0078] In step S2, template amplification is performed using the RPA constant temperature amplification method. The RPA amplification reagent is purchased from Anpu Future Biotechnology Co., Ltd., wherein RPA amplification reaction freeze-dried buffer balls, RPA freeze-dried enzyme Mix balls, 2 μL each of forward and reverse primers (10 uM), 0.5 μL of nfo probe (10 uM), and 47.5 μL of the DNA template of the sample to be tested are added to the reaction tube and mixed evenly. After the tube cap is closed, centrifugation is performed instantly to start the RPA reaction. The entire reaction is between 37-42°C and the reaction time is 10-30 min.

[0079] Step S3 specifically includes:

[0080] Take 10μL of amplification product and mix it with 190μL of distilled water, immerse the lateral flow chromatography test strip in the mixture, observe the control line and the test line, and take out the test strip 2 minutes after the liquid reaches the top of the test strip. The test results show that both the test line and the quality control line have color bands: This kit can detect Helicobacter pylori contained in dental plaque, which means that it can meet actual needs.

[0081] The experimental results of the above examples show that the rapid detection kit for Helicobacter pylori (constant temperature amplification test strip method) provided by the present invention has strong specificity, high sensitivity, and convenient operation, and can realize rapid detection of Helicobacter pylori by visual observation.

[0082] The above embodiments are only preferred embodiments of the present invention and should not be used to limit the protection scope of the present invention.

[0083] Any changes or modifications that are made to the main design concept and spirit of the present invention without any substantial significance, and the technical problems they solve are still consistent with the present invention, shall be included in the protection scope of the present invention. The present invention can be used for auxiliary diagnosis of Helicobacter pylori infection, and the test results are for reference only and cannot be used alone as a basis for confirming or excluding cases.

Claims

1. A primer probe set for rapid detection of Helicobacter pylori, characterized in that: The primer probe set comprises a Helicobacter pylori forward primer, a Helicobacter pylori reverse primer and a Helicobacter pylori probe; The nucleotide sequence of the Helicobacter pylori forward primer is shown in SEQ ID No. 1; The nucleotide sequence of the Helicobacter pylori reverse primer is shown in SEQ ID No. 2; The nucleotide sequence of the Helicobacter pylori probe is shown in SEQ ID No.

3.

2. The primer probe set according to claim 1, characterized in that: The 5' end of the Helicobacter pylori reverse primer is labeled with biotin.

3. The primer probe set according to claim 1, characterized in that: The 5' end of the Helicobacter pylori probe is modified with an antigenic molecule marker; the 3' end of the Helicobacter pylori probe is modified with a polymerase extension blocking group C3-Spacer; and the middle 18 bases away from the 3' end are treated with tetrahydrofuran THF residues.

4. The primer probe set according to claim 3, characterized in that: The antigenic molecule marker can be biotin, tetramethylrhodamine, digoxigenin, FAM, FITC, and FAM is more preferred.

5. A kit for rapid detection of Helicobacter pylori, characterized in that: It comprises a primer probe set for amplifying the DNA of the sample to be tested; the primer probe set for amplifying the DNA of the sample to be tested is the primer probe set or its complementary chain as claimed in claim 1, or has a single sequence homology of 50%, 60%, 70%, 80%, 90%, 95%, 99% or more to the sequence as claimed in claim 1 or its complementary chain; further preferably, it comprises the primer probe set as claimed in claim 1.

6. A kit for rapid detection of Helicobacter pylori according to claim 5, characterized in that: The kit comprises a nucleic acid extraction reagent, an RPA amplification reaction freeze-dried buffer ball, an RPA freeze-dried enzyme Mix ball and a lateral flow chromatography test strip; The lateral flow chromatography test strip comprises a sample loading pad area, a colloidal gold labeled antibody area, a nitrocellulose membrane layer, and a reaction chromatography membrane absorption area; the nitrocellulose membrane layer is provided with a detection line sprayed with streptavidin and a quality control line sprayed with antibodies; the colloidal gold labeled antibody area is provided with colloidal gold labeled anti-FAM antibodies.

7. A kit for rapid detection of Helicobacter pylori according to claim 6, characterized in that: The RPA amplification reaction freeze-dried buffer ball comprises the following components: polyethylene glycol 1.5-5wt%; Tris 10-50mM; sodium acetate 200-240mM; dithiothreitol 1-10mM; ATP 1.5-3.5mM; creatine phosphate disodium salt 20-75mM; phosphokinase 10-200ng / μL; deoxyribonucleoside triphosphate, 450-500μM; trehalose 2.5-7.5wt%; mannitol 40-50ng / μL; activator magnesium acetate; the RPA freeze-dried enzyme Mix ball comprises a recombinase, a single-stranded DNA binding protein, a DNA polymerase and a nuclease endonuclease.

8. A kit for rapid detection of Helicobacter pylori according to claim 7, characterized in that: The amplification temperature is 37-42°C and the amplification time is 15-20 minutes.

9. A method for using the kit for rapid detection of Helicobacter pylori as claimed in claim 6, characterized in that: The method comprises the following steps: using the primer probe set of claim 1 to amplify the DNA of the sample to be tested, and then using a lateral flow chromatography test strip to detect, if the test line shows a band, the sample to be tested contains Helicobacter pylori, and if no band is shown, it does not contain Helicobacter pylori.

10. The method according to claim 9, characterized in that The sensitivity of this method for the DNA of the sample to be tested is 50 copies / T.

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