CN118064614A - Primer probe group for helicobacter pylori ERA detection and detection method - Google Patents
Primer probe group for helicobacter pylori ERA detection and detection method Download PDFInfo
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Abstract
The invention discloses a primer probe group for detecting helicobacter pylori ERA and a detection method, and belongs to the technical field of biological detection. The invention designs a specific primer pair and a probe aiming at 16s rRNA of helicobacter pylori, and is matched with a corresponding dissolving agent, an activating agent, a detection test strip and a reaction system, so that helicobacter pylori in a sample can be detected rapidly. The detection method is simple and convenient to operate, and large complex and expensive instruments are not needed; the result identification is visual and convenient, and the processes of nucleic acid electrophoresis and the like are not needed; the method is quick and efficient, and the result can be judged in a short time; the specificity and the sensibility are good, the bacillus can be distinguished from common intestinal bacillus, and the concentration of the detected crude Hp genome DNA with the lowest sensibility is 0.1 ng/. Mu.L; has important significance for establishing a rapid detection method of helicobacter pylori.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a primer probe group for detecting helicobacter pylori ERA and a detection method.
Background
Helicobacter pylori (Helicobacterpylori, hp) is a gram-negative and transmissible pathogen, and national epidemiological data on Hp infection is not optimistic. Hp infection is closely related to diffuse gastric cancer, intestinal gastric cancer, gastric mucosa-associated lymphoid tissue lymphoma. In addition, helicobacter pylori is closely related to the onset of diseases such as iron deficiency anemia, periodontal disease, skin disease, idiopathic thrombocytopenic purpura, even inappropriate coronary heart disease, hypertension, and the like. According to the latest expert consensus on helicobacter pylori infection in China for the sixth time, all Hp positive patients are treated as necessary, and the Hp eradication indication proves that helicobacter pylori infection can be eradicated. Comprehensive current researches on cost benefits of Hp eradication, gastric cancer and other diseases, accurate diagnosis of helicobacter pylori has important positive significance on curing Hp-induced diseases.
At present, the detection method of helicobacter pylori mainly comprises an endoscope examination, a rapid urease experiment (RUT), an invasive test such as histology and bacterial culture, a urease expiration experiment (UBL), fecal antigen detection, a non-invasive test such as serology and a molecular diagnosis method. UBT experiments are considered as safe, effective, highly accurate non-invasive experiments, but the administration of bismuth agents, PPI, antibiotics, antibacterial agents can affect the judgment of UBT outcome; meanwhile, a plurality of researches prove that strains such as klebsiella pneumoniae and proteus mirabilis which produce urease in the oral cavity of children can cause false positive of the result; antigen detection can be affected by several factors: the unformed or watery stool specimens can reduce the accuracy of the results; patients with upper gastrointestinal bleeding also develop false negative results; fecal specimens and/or their suspensions can rapidly fail in high temperature environments. The differences in helicobacter pylori antigenicity and in individual genetics affect the level of antibody secretion, and the inability to distinguish between acute and past infections, results in serological tests that are still not sufficiently specific and sensitive; the PCR detection technique requires a special laboratory, relies on talents with specialized operating techniques, and special instrumentation, resulting in limited application.
The enzymatic recombination isothermal amplification technology (ERA technology) is developed by Souzhou first reach gene technology Co., ltd.s.A. of China, by simulating the principle of self amplification and replication of organism genetic material, a special amplification reaction system is established by modifying a multienzyme system such as recombinase, exonuclease, polymerase and the like through genetic engineering, and the specific segment of trace DNA/RNA can be amplified by billions times within minutes under the constant temperature condition of 37-42 ℃, so that the DNA/RNA detection can more easily meet the requirements of low-temperature, rapid and household detection. Therefore, the research on the method and the system which can be used for the routine and rapid detection of the helicobacter pylori based on the ERA technology has wide prospect.
Disclosure of Invention
The invention aims to provide a primer probe group for detecting helicobacter pylori ERA and a detection method, which are used for detecting helicobacter pylori based on ERA technology, can obviously shorten the detection time of helicobacter pylori, can carry out integral amplification and detection at a lower temperature, have high specificity and sensitivity, and simultaneously combine with a lateral flow detection test strip to quickly and intuitively observe an amplification result, thereby providing a potential effective means for quick detection of helicobacter pylori.
The invention provides a primer probe group for helicobacter pylori ERA detection, which comprises a forward primer Hp-F with a nucleotide sequence shown as SEQ ID No.1, a reverse primer Hp-R with a nucleotide sequence shown as SEQ ID No.2 and a probe Hp-P with a nucleotide sequence shown as SEQ ID No. 3.
Preferably, biotin groups are labeled at the 5' end of the reverse primer Hp-R;
A fluorescent group is marked at the 5-end of the probe Hp-P, and a modification group for suppressing polymerase extension or amplification is marked at the 3-end of the probe Hp-P.
The invention also provides a helicobacter pylori ERA detection method for non-disease diagnosis and treatment purposes, which comprises the steps of preparing a reaction system from a DNA template and a dissolving agent of a target detection object and the primer probe group for ERA detection, carrying out ERA amplification, and carrying out color development and observation on a reaction product by utilizing a matched lateral flow detection test strip, wherein the amplification is positive when obvious strips appear on a C line and a T line.
Preferably, the reaction system comprises, in 48 μl: 2. Mu.L of DNA template, 20. Mu.L of dissolving agent, 2.1. Mu.L of forward primer Hp-F, 2.1. Mu.L of reverse primer Hp-R, 0.6. Mu.L of probe Hp-P and the balance of ddH 2 O.
Preferably, the reaction system is configured in freeze-dried amplification reagent tubes, each tube further comprising 2 μl of activator.
Preferably, the ERA amplification is carried out at 37℃for 15min.
The invention also provides a helicobacter pylori ERA detection kit which comprises the primer probe group for ERA detection, a dissolving agent and an activating agent.
Preferably, the kit further comprises a freeze-dried amplification reagent tube and a corresponding lateral flow detection test strip.
The beneficial effects are that:
the invention provides a primer probe group for detecting helicobacter pylori ERA, which is used for designing a specific primer pair and a probe aiming at 16s rRNA of helicobacter pylori, matching with a corresponding dissolving agent, an activating agent and a detection test strip, and a reaction system, and can be used for rapidly detecting helicobacter pylori in a sample. The detection method is simple and convenient to operate, and large complex and expensive instruments are not needed; the result identification is visual and convenient, and the processes of nucleic acid electrophoresis and the like are not needed; the method is quick and efficient, and the result can be judged in a short time; the specificity and the sensibility are good, the bacillus can be distinguished from common intestinal bacillus, and the concentration of the detected crude Hp genome DNA with the lowest sensibility is 0.1 ng/. Mu.L; has important significance for establishing a rapid detection method of helicobacter pylori.
Drawings
FIG. 1 is a diagram showing the result of specific detection of helicobacter pylori by the primer-probe set designed in the present invention, wherein NTC is a negative control;
FIG. 2 is a graph showing the results of the specificity of the ERA test strip detection system for helicobacter pylori, wherein the templates used are genomic DNA of helicobacter pylori (H.pyri), proteus mirabilis (P.mirabilis), escherichia coli (E.coli) and Salmonella typhi (S.typhosa), respectively;
FIG. 3 is a graph of temperature optimization results of ERA amplification conditions against helicobacter pylori, wherein the temperature gradient is 25 ℃,30 ℃,37 ℃,40 ℃ and 55 ℃;
FIG. 4 is a graph of the results of time optimization of ERA amplification conditions against H.pylori, wherein the time gradients are 0min,5min,10min,15min,20min and 30min;
FIG. 5 is a graph showing the sensitivity results of an ERA test strip detection system for helicobacter pylori, wherein the concentration of crude extract genomic DNA of helicobacter pylori is 0 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 10 -1ng/μL,10-2ng/μL,10-3 ng/. Mu.L and 10 -4 ng/. Mu.L, respectively;
FIG. 6 is a graph showing the detection efficiency of helicobacter pylori ERA test strip for detecting a positive sample of helicobacter pylori in clinic.
Detailed Description
The invention provides a primer probe group for helicobacter pylori ERA detection, which comprises a forward primer Hp-F with a nucleotide sequence shown as SEQ ID No.1, a reverse primer Hp-R with a nucleotide sequence shown as SEQ ID No.2 and a probe Hp-P with a nucleotide sequence shown as SEQ ID No. 3.
The 5 ' end of the reverse primer Hp-R in the present invention is preferably labeled with a biotin group, the 5 ' end of the probe Hp-P is labeled with a fluorescent group, the 3 ' end is labeled with a modification group for suppressing extension or amplification of polymerase, and in the examples, the fluorescent group FITC is exemplified, but it cannot be considered as the whole protection scope of the present invention.
Hp-F：TATCTAACGAATAAGCACCGGCTAACTCCG；
Hp-R：biotin-CCTTTGAGTTTTAATCTTGCGACCGTACT；
Hp-P：FITC-TCCGTAAGGAGGAGGAAGGTGGGGACGACGT[THF]AAGT CATCATGGCCCT-SpC3。
The primer probe set of the invention preferably uses helicobacter pylori 16s rRNA gene as a detection target gene, and designs and synthesizes a special primer pair and a probe based on ERA and test strip detection.
The invention also provides a method for detecting helicobacter pylori ERA for non-diagnosis and treatment, which comprises the steps of preparing a reaction system from a DNA template and a dissolving agent of a target detection object and the primer probe group for ERA detection, carrying out ERA amplification, and carrying out color development and observation on a reaction product by utilizing a matched lateral flow detection test strip, wherein the amplification is positive when red strips appear on a C line and a T line.
The establishment of the method can rapidly detect helicobacter pylori, which prompts a patient to have helicobacter pylori infection, and the helicobacter pylori infection is an influencing factor of occurrence of gastritis and gastric cancer, therefore, the establishment of the method aims at detecting helicobacter pylori, provides a basis for diagnosing helicobacter pylori patients and treatment thereof as an intermediate result, and belongs to detection for non-diagnosis and treatment purposes.
The reaction system of the present invention preferably comprises, in terms of 48. Mu.L: 2. Mu.L of DNA template, 20. Mu.L of dissolving agent, 2.1. Mu.L of forward primer Hp-F, 2.1. Mu.L of reverse primer Hp-R, 0.6. Mu.L of probe Hp-P and the balance of ddH 2 O. In the present invention, it is preferable to arrange the reaction system in a freeze-dried amplification reagent tube, and 2. Mu.L of an activator is further contained in each tube.
Preferably, the ERA amplification is accomplished by incubation at 37℃for 15min after a brief centrifugation after configuration. After ERA reaction is finished, 5 mu L of detection reaction products are taken, 245 mu L of pure water is added for diluting a sample, then a prefabricated test strip (TS 101 of Suzhou first gene company) is inserted into the diluted products, and the result is interpreted after waiting for 2-3 minutes; the positive was marked by the presence of distinct bands on both the C and T lines. The dissolvent and the activator are the self-contained content of KS105 kit of Suzhou first reach gene company.
The invention also provides a helicobacter pylori ERA detection kit which comprises the primer probe group for ERA detection, a dissolving agent and an activating agent.
The kit of the invention preferably further comprises a freeze-dried amplification reagent tube and a corresponding lateral flow detection test strip.
In order to further illustrate the present invention, a primer probe set for detecting helicobacter pylori ERA and a detection method provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Primer pair and probe design and verification for ERA test paper type detection of targeting helicobacter pylori 16s rRNA
Primer pair and probe design: the sequence of H.pylori 16s rRNA was determined by querying the NCBI database (https:// www.ncbi.nlm.nih.gov). Designing a probe and a primer pair (SEQ ID No. 1-SEQ ID No. 3) by utilizing PRIMERPRIMER 6.0.0 software according to the design principle of the probe and the primer of the ERA reaction, and simultaneously analyzing the quality of the primer pair and the probe;
extraction of helicobacter pylori genomic DNA: 1mL of 0.5 M.H.pylori (ATCC 26695) culture was boiled at 100℃for 10min, and the genomic DNA was extracted sufficiently.
(3) Preliminary verification of feasibility and specificity of primer pairs and probes: the genomic DNA is used as a template, and a negative blank control is arranged at the same time, and the feasibility and the specificity of the genome DNA are verified through ERA reaction and test strip detection. The method comprises the following specific steps:
After preparing an ERA amplification reaction system using a lytic reagent, a primer pair and a probe, adding the reaction system to a freeze-dried amplification reagent tube (Suzhou Mida Gene Co., KS 105), adding 2. Mu.L of an activator (Suzhou Mida Gene Co., KS 105), and after a short centrifugation, incubating at 37℃for 15min. After the reaction is completed, 5 mu L of detection reaction products are taken, 245 mu L of pure water is added for diluting a sample, and then a prefabricated test strip (TS 101 of Suzhou first reach gene company) is inserted into the diluted products for waiting 2-3 minutes, and then the result is interpreted; the positive bands were seen on both lines C and T.
48 Μl system: 2. Mu.L of DNA template, 20. Mu.L of lytic reagent, 2.1. Mu.L of forward primer Hp-F (10. Mu.M), 2.1. Mu.L of reverse primer Hp-R (10. Mu.M), 0.6. Mu.L of probe Hp-P (10. Mu.M) and the balance ddH 2 O.
As a result, as shown in FIG. 1, the detection system was successful in amplifying the 16s rRNA target sequence of H.pylori and showed a distinct positive band, compared to the negative control. As described above, a set of forward primer (Hp-F), reverse primer (Hp-R) and probe (Hp-P) was finally obtained. Wherein the 5 ' end of Hp-R is labeled with biotin group (biotin), the 5 ' end of Hp-P is labeled with fluorescent group (FITC), and the 3 ' end is labeled with a modification group (C3-SPACER) which suppresses polymerase extension or amplification.
Example 2
Specific verification of helicobacter pylori ERA test strip detection system
Salmonella typhi (CICC 10867), proteus mirabilis (ATCC 35659), escherichia coli (ATCC 25922) were cultured separately, and genomic DNA was extracted by boiling. A negative control was set up and the four strains were tested separately according to the ERA reaction system and test procedure described in example 1.
As shown in FIG. 2, the primer pair and the probe can only positively amplify helicobacter pylori, and other common intestinal bacteria are negative. The primer pair and the probe set do not cross react with other pathogens, and have better specificity.
Example 3
Reaction condition optimization of helicobacter pylori ERA test strip detection system
The helicobacter pylori ERA test strip detection system is optimized from ERA reaction temperature and reaction time respectively.
Optimization of ERA reaction temperature: a corresponding ERA reaction system was prepared as described in example 1, and was added to a freeze-dried amplification reagent tube (St. John's gene, KS 105) and 2. Mu.L of an activator (St. John's gene, KS 105), and after brief centrifugation, the ERA reaction system was reacted at 25℃at 30℃at 37℃at 40℃and at 55℃for 15 minutes, respectively, and then the optimum temperature of the ERA reaction was screened using a test strip to display the results. As shown in FIG. 3, the amplification yield tended to saturate at 37℃and the bands showed the most remarkable, so that the optimum temperature of the reaction system was determined to be 37 ℃.
Optimization of ERA reaction time: the corresponding ERA reaction system was prepared as described in example 1, added to a freeze-dried amplification reagent tube (St. Johnst Gene Co., st. Johnst., KS 105), and 2uL of an activator (St. Johnst Gene Co., st. Johnst., KS 105) was added, and after a short centrifugation, the ERA reaction system was subjected to respective reactions at 37℃for 0min, 5min, 10min, 15min, 20min and 30min, and then ERA reaction times were screened using a test strip to display the results. As shown in FIG. 4, the band showed that the reaction time was 5min, and therefore, it was confirmed that the reaction system could be used for 5min as ERA reaction time later.
Example 4
Sensitive detection of helicobacter pylori ERA test strip detection system
To determine the sensitivity of the H.pylori ERA test strip detection system of examples 1 to 3 (wherein the reaction temperature was 37℃and the reaction time was 5 min), H.pylori genomic DNA was roughly extracted by boiling, the concentration thereof was adjusted to 10ng/uL, and the minimum detection limit of this detection system was determined by using the test strip display result after placing the ERA reaction system at 37℃for 5min with 10-fold dilutions (0 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 10 -1ng/μL,10-2ng/μL,10-3ng/μL,10-4 ng/. Mu.L) of H.pylori genomic DNA as a template, the corresponding ERA reaction system was prepared as described in example 1, and added to a freeze-dried amplification reagent tube (St. Arrive gene, KS 105) and 2. Mu.L of an activator (St. Arrive gene, KS 105) was added. As shown in FIG. 5, the detection result shows that the reaction system can detect helicobacter pylori genomic DNA with an initial concentration of 10 -1 ng/. Mu.L (final concentration of 4 pg/. Mu.L), and has better sensitivity.
Example 5
Detection of clinical helicobacter pylori positive sample by helicobacter pylori ERA test strip
To determine the potential for clinical use of the H.pylori ERA test strip detection system of examples 1-3 (wherein the reaction temperature was 37℃and the reaction time was 5 min), 20 cases of clinical sample genomic DNA confirmed by urease experiments and real-time fluorescent quantitative PCR for H.pylori infection were roughly extracted as templates, the corresponding ERA reaction system was prepared as described in example 1, and was added to a freeze-dried amplification reagent tube (St. John, KS 105) and 2. Mu.L of an activator (St. John, KS 105) and after brief centrifugation, the ERA reaction system was subjected to a reaction at 37℃for 5min, and the detection results were displayed using the test strip. As shown in FIG. 6, the reaction system can accurately detect 17 samples in 20 clinical positive samples, and the positive prediction rate reaches 85%.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (8)
1. A primer probe group for detecting helicobacter pylori by utilizing an enzymatic recombination isothermal amplification method is characterized by comprising a forward primer Hp-F with a nucleotide sequence shown as SEQ ID No.1, a reverse primer Hp-R with a nucleotide sequence shown as SEQ ID No.2 and a probe Hp-P with a nucleotide sequence shown as SEQ ID No. 3.
2. The primer probe set of claim 1, wherein a biotin group is labeled at the 5' end of the reverse primer Hp-R;
A fluorescent group is marked at the 5-terminus of the probe Hp-P, and a modification group for suppressing polymerase extension or amplification is marked at the 3-terminus.
3. A method for detecting helicobacter pylori by utilizing an enzymatic recombination isothermal amplification method for non-disease diagnosis and treatment purpose is characterized by comprising the steps of preparing a reaction system by a DNA template and a dissolving agent of a target detection object and a primer probe set according to claim 1 or 2, performing enzymatic recombination isothermal amplification, and developing and observing an amplification product by utilizing a matched lateral flow detection test strip, wherein amplification is positive when obvious strips appear on a C line and a T line.
4. A method according to claim 3, wherein the reaction system comprises, in 48 μl: 2. Mu.L of DNA template, 20. Mu.L of dissolving agent, 2.1. Mu.L of forward primer Hp-F, 2.1. Mu.L of reverse primer Hp-R, 0.6. Mu.L of probe Hp-P and the balance of ddH 2 O.
5. The method of claim 4, wherein the reaction system is disposed in a lyophilized amplification reagent tube, each tube further comprising 2 μl of activator.
6. A method according to claim 3, wherein the ERA amplification is carried out at a temperature of 37 ℃ for a period of 15 minutes.
7. A kit for detecting helicobacter pylori ERA, which comprises the primer probe set for detecting ERA according to claim 1 or 2, a dissolving agent and an activating agent.
8. The helicobacter pylori ERA detection kit according to claim 7, further comprising a lyophilized amplification reagent tube and a corresponding lateral flow detection test strip.
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