CN107365851B - LAMP (loop-mediated isothermal amplification) rapid detection method for helicobacter pylori - Google Patents

LAMP (loop-mediated isothermal amplification) rapid detection method for helicobacter pylori Download PDF

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CN107365851B
CN107365851B CN201710691336.0A CN201710691336A CN107365851B CN 107365851 B CN107365851 B CN 107365851B CN 201710691336 A CN201710691336 A CN 201710691336A CN 107365851 B CN107365851 B CN 107365851B
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CN107365851A (en
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冯洁
谢建芸
高诚
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SHANGHAI LAB ANIMAL RESEARCH CENTER
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Abstract

The invention provides a detection primer group of helicobacter pylori LAMP, which comprises two outer primers, two inner primers and a loop primer, wherein the primers have nucleotide sequences of SEQ ID NO. 1-NO 5. The invention also provides a PCR detection system of helicobacter pylori LAMP, which comprises the following components: a primer group: two outer primers, two inner primers and a loop primer; 2 times reaction buffer solution, DNA polymerase, fluorescent dye, DNA template and sterilized purified water. The invention discloses a LAMP primer and an amplification method for specifically detecting helicobacter pylori, which are not limited by culture conditions, have the advantages of simple operation, good specificity, high sensitivity, easy observation of results, low requirement on instruments and equipment and the like, can be used for quickly detecting the helicobacter pylori, and are suitable for popularization and application.

Description

LAMP (loop-mediated isothermal amplification) rapid detection method for helicobacter pylori
Technical Field
The invention belongs to the field of biochemical detection, and particularly relates to a rapid LAMP detection method for helicobacter pylori.
Background
Helicobacter pylori (helicobacter pylori) is an important pathogen of zoonosis and can cause various diseases such as chronic active hepatitis, liver cancer, breast cancer, appendicitis, colitis and the like; the helicobacter pylori is a microaerophilic bacterium, has high nutritional requirement, is tedious in biochemical identification test operation, consumes time and labor, can obtain a detection result in 2-3 weeks generally, and has very low sensitivity. Usually resulting in missed detection and therefore culture methods are not suitable for rapid detection. Serological methods are simple to operate and can detect a large number of samples at the same time, but because of the existence of cross-reactions between different helicobacter species, false positives are possible, making the species-specific identification of helicobacter hepaticus difficult. The PCR method has the advantages of rapidness, specificity and sensitivity, and is widely applied, but the detection process needs expensive equipment such as an amplification instrument and the like for support, and the problem of false positive caused by pollution is easy to occur, so that the popularization and the application of the method on the first-line site are limited.
Disclosure of Invention
In view of the above, the present invention aims to provide a primer set for LAMP detection of helicobacter pylori, which comprises two outer primers, two inner primers and a loop primer, wherein the primers have the following nucleotide sequences: f3 forward outer primer: 5'-GCAGCTTTAACATTAACTTGTGTA-3' (SEQ ID NO. 1); b3 reverse outer primer: 5'-AATGCGAATGTCGCTCAA-3' (SEQ ID NO. 2);
FIP forward inner primer:
5’-GCCCAGCTTGATAAACTCCGTATAGTTGAAACAAGTTGAATCTGTG-3’(SEQ ID NO.3)；
BIP reverse inner primer:
5’-TCTGCCATATCCATAACTACCATTGATCTCCATTTTGATGGTATCG-3’；(SEQ ID NO.4)
LB reverse loop primer: 5'-CCTTTAAGGCTTGTAACCCCA-3' (SEQ ID NO. 5).
Preferably, the LAMP PCR detection system for helicobacter pylori comprises the following components: a primer group: two outer primers, two inner primers and a loop primer; 2 times reaction buffer solution, DNA polymerase, fluorescent dye, DNA template and sterilized purified water.
Preferably, in the PCR detection system of helicobacter pylori LAMP of the present invention, the primers have the following nucleotide sequences:
f3 forward outer primer: 5'-GCAGCTTTAACATTAACTTGTGTA-3' (SEQ ID NO. 1); b3 reverse outer primer: 5'-AATGCGAATGTCGCTCAA-3' (SEQ ID NO. 2);
FIP forward inner primer:
5’-GCCCAGCTTGATAAACTCCGTATAGTTGAAACAAGTTGAATCTGTG-3’(SEQ ID NO.3)；
BIP reverse inner primer:
5’-TCTGCCATATCCATAACTACCATTGATCTCCATTTTGATGGTATCG-3’；(SEQ ID NO.4)。
preferably, in the PCR detection system of helicobacter pylori LAMP of the present invention, the PCR detection system includes: 1 μ L of each of 5pM outer primers F3 and B3 in a 25 μ L LAMP PCR reaction system; 1 mu L of each of 40pM of inner primers FIP and BIP; reverse loop primer LB of 20pM 1. mu.L; 2 × 12.5 μ L of reaction buffer; bst DNA polymerase 1 u L; fluorescent dye 1 μ L; DNA template 2. mu.L, the remainder was made up with sterile purified water.
Another objective of the invention is to provide a rapid detection method of helicobacter pylori LAMP, which comprises the following steps:
1) extracting helicobacter DNA from the sample;
2) the helicobacter DNA is taken as a template, and the primer group is put into an isothermal amplification instrument for PCR amplification;
3) the amplification results were analyzed visually or by detection on 2.0% agarose gel electrophoresis.
Preferably, in the rapid detection method of helicobacter pylori LAMP of the present invention, the PCR system comprises a primer set; and 2 times reaction buffer solution, DNA polymerase, fluorescent dye, DNA template and sterilized purified water.
Preferably, in the rapid detection method of helicobacter pylori LAMP of the present invention, the PCR detection system comprises: 1 μ L of each of 5pM outer primers F3 and B3 in a 25 μ L LAMP PCR reaction system; 1 mu L of each of 40pM of inner primers FIP and BIP; reverse loop primer LB of 20pM 1. mu.L; 2 × 12.5 μ L of reaction buffer; bst DNA polymerase 1 u L; fluorescent dye 1 μ L; DNA template 2. mu.L, the remainder was made up with sterile purified water.
Preferably, in the rapid detection method of helicobacter pylori LAMP of the present invention, the visual inspection method is that after the LAMP reaction is finished, green fluorescence is observed in the color development result, and the result is judged to be positive, and orange is judged to be negative.
Preferably, in the rapid detection method of helicobacter pylori LAMP of the present invention, the agarose gel electrophoresis method: and detecting 2 microliter of amplification product by 2% agarose gel electrophoresis, and judging the amplification product to be positive if a ladder-shaped band characteristic to LAMP appears, and judging the amplification product to be negative if no amplification band appears.
The invention also aims to provide a kit for rapidly detecting the LAMP of the helicobacter pylori, which comprises the primer group or the PCR detection system.
The invention discloses a LAMP primer and an amplification method for specifically detecting helicobacter pylori, which are not limited by culture conditions, can finish the detection of the helicobacter pylori within 60min only by adding samples according to an established system, have the advantages of simple operation, good specificity, high sensitivity, easy observation of results, low requirement on instruments and equipment and the like, can be used for quickly detecting the helicobacter pylori, and are suitable for popularization and application.
Drawings
FIG. 1 is a LAMP amplification chart for a specificity test in one embodiment of the present invention;
FIG. 2 is a graph showing the results of visual detection of the specificity test in one embodiment of the present invention;
FIG. 3 is a diagram showing the results of electrophoresis detection in a specificity test according to an embodiment of the present invention;
FIG. 4 is a graph of the results of a visual test of a susceptibility test in accordance with an embodiment of the invention;
FIG. 5 is a diagram showing the results of electrophoresis detection of the sensitivity test in one embodiment of the present invention.
Detailed Description
The strain information used in the present invention is as follows: helicobacter hepatica ATCC 51449, helicobacter bilis ATCC51630, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 27853, Pasteurella pneumophila ATCC35149, Citrobacter rodent ATCC 51116, and Corynebacterium bovis ATCC 7715 were all purchased from the American Standard Biotech Collection. Klebsiella pneumoniae CMCC46117, Salmonella typhimurium CMCC50115, Shigella flexneri CMCC 51572 and Alcaligenes faecalis CMCC 40001 are all purchased from China medical microorganism strain preservation management center.
Deoxyribonucleic acid amplification kit (loop-mediated isothermal amplification method) (purchased from Rongyan Biotechnology Ltd.), and bacterial genomic DNA extraction kit (purchased from Tiangen Biochemical technology Ltd.).
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 LAMP detection primer design for helicobacter pylori
F3 forward outer primer: 5'-GCAGCTTTAACATTAACTTGTGTA-3' (SEQ ID NO.1)
B3 reverse outer primer: 5'-AATGCGAATGTCGCTCAA-3' (SEQ ID NO.2)
FIP forward inner primer: 5'-GCCCAGCTTGATAAACTCCGTATAGTTGAAACAAGTTGAATCTGTG-3' (SEQ ID NO.3)
BIP reverse inner primer: 5'-TCTGCCATATCCATAACTACCATTGATCTCCATTTTGATGGTATCG-3'
(SEQ ID NO.4)
LB reverse loop primer: 5'-CCTTTAAGGCTTGTAACCCCA-3' (SEQ ID NO.5)
Example 2 genomic DNA extraction, LAMP amplification System establishment and results
1. Extracting the genomic DNA of the helicobacter pylori by using a TIANGEN bacterial genomic DNA extraction kit, measuring the OD260/280 by using an ultraviolet spectrophotometer to be in the range of 1.6-2.0, and storing at the temperature of-20 ℃ for later use.
The LAMP reaction system is as follows:
Figure BDA0001377962420000041
and (3) putting the established LAMP amplification system into an isothermal amplification instrument, and reacting for 45min at 63 ℃. The amplification results were analyzed visually or by detection on 2.0% agarose gel electrophoresis.
Respectively carrying out LAMP amplification by using helicobacter hepatica, helicobacter biliary tract, staphylococcus aureus, pseudomonas aeruginosa, pasteurella pneumophila, rodent bacillus citrate, corynebacterium bovis, klebsiella pneumoniae, salmonella typhimurium, shigella flexneri, alcaligenes faecalis and water as templates, and observing the amplification result, wherein the result is shown in figures 1-3, only specific amplification of the helicobacter hepatica occurs, and amplification does not occur in other templates; FIG. 2 shows that only helicobacter hepaticus exhibits green fluorescence (in the reference numerals of FIG. 2, test tubes 1 to 12 are 1 helicobacter hepaticum, 2: helicobacter biliary, 3: Staphylococcus aureus, 4: Pseudomonas aeruginosa, 5: Pasteurella pneumophila, 6: Citrobacter rodent, 7: Corynebacterium bovis, 8: Klebsiella pneumoniae, 9: Salmonella typhimurium, 10: Shigella flexneri, 11: Alcaligenes faecalis, 12: water); FIG. 3 shows that only helicobacter hepaticus appears in a specific band, which indicates that the LAMP detection method established in the present invention has good specificity (in FIG. 3, the lane labels are M: DL 2000 DNAmarker; 1: helicobacter hepaticus; 2: helicobacter biliary tract; 3: Staphylococcus aureus; 4: Pseudomonas aeruginosa; 5: Pasteurella pneumophila; 6: Citrobacter rodent; 7: Corynebacterium bovis; 8: Klebsiella pneumoniae; 9: Salmonella typhimurium; 10: Shigella flexneri; 11: Alcaligenes faecalis; 12: water, respectively).
Example 3: LAMP sensitivity assay
The genomic DNA of the helicobacter pylori extracted in example 2 is subjected to gradient dilution by 10 times, and the DNA subjected to gradient dilution is used as a template, and the LAMP detection method established by the invention is adopted for amplification so as to verify the sensitivity of the LAMP system established by the invention.
The results of the visual observation are shown in fig. 4, in which green fluorescence appears in the test tubes 1 to 9, green fluorescence does not appear in the test tubes 10 and 11, and the concentrations in the test tubes are 1: 4.3X 109copy/uL; 2: 4.3X 108copy/uL; 3: 4.3X 107copy/uL; 4: 4.3X 106copy/uL; 5: 4.3X 105copy/uL; 6: 4.3X 104copy/uL; 7: 4.3X 103copy/uL; 8: 4.3X 102copy/uL; 9: 4.3X 101copy/uL; 10: 4.3X 100copy/uL; 11: 4.3X 10-1copy/uL.
The 2% agarose electrophoresis is shown in FIG. 5, wherein the specific bands are shown in lanes 1-9, and no specific band is shown in lanes 10 and 11, wherein the bands are M: DL 2000 DNAmarker; 1: 4.3X 109copy/uL; 2: 4.3X 108copy/uL; 3: 4.3X 107copy/uL; 4: 4.3X 106copy/uL; 5: 4.3X 105copy/uL; 6: 4.3X 104copy/uL; 7: 4.3X 103copy/uL; 8: 4.3X 102copy/uL; 9: 4.3X 101copy/uL; 10: 4.3X 100copy/uL; 11: 4.3X 10-1copy/uL.
The results of FIGS. 4-5 show that the lowest detection limit of the LAMP detection method established by the invention is 43 copies/uL.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<120> LAMP rapid detection method for helicobacter pylori
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aatgcgaatg tcgctcaa 18
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gcccagcttg ataaactccg tatagttgaa acaagttgaa tctgtg 46
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tctgccatat ccataactac cattgatctc cattttgatg gtatcg 46
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cctttaaggc ttgtaacccc a 21

Claims (4)

1. A detection primer group of helicobacter pylori LAMP comprises two outer primers, two inner primers and a loop primer, wherein the nucleotide sequence of the primers is as follows:
f3 forward outer primer: 5'-GCAGCTTTAACATTAACTTGTGTA-3' (SEQ ID NO. 1);
b3 reverse outer primer: 5'-AATGCGAATGTCGCTCAA-3' (SEQ ID NO. 2);
FIP forward inner primer:
5’-GCCCAGCTTGATAAACTCCGTATAGTTGAAACAAGTTGAATCTGTG-3’(SEQ ID NO.3)；
BIP reverse inner primer:
5’-TCTGCCATATCCATAACTACCATTGATCTCCATTTTGATGGTATCG-3’；(SEQ ID NO.4)
LB reverse loop primer: 5'-CCTTTAAGGCTTGTAACCCCA-3' (SEQ ID NO. 5).
2. A LAMP detection system for helicobacter pylori comprises the following components: a primer group: two outer primers, two inner primers and a loop primer; 2 times of reaction buffer solution, DNA polymerase, fluorescent dye, DNA template and sterilized purified water;
the primer consists of the following nucleotide sequences:
f3 forward outer primer: 5'-GCAGCTTTAACATTAACTTGTGTA-3' (SEQ ID NO. 1);
b3 reverse outer primer: 5'-AATGCGAATGTCGCTCAA-3' (SEQ ID NO. 2);
FIP forward inner primer:
5’-GCCCAGCTTGATAAACTCCGTATAGTTGAAACAAGTTGAATCTGTG-3’(SEQ ID NO.3)；
BIP reverse inner primer:
5’-TCTGCCATATCCATAACTACCATTGATCTCCATTTTGATGGTATCG-3’；(SEQ ID NO.4)；
LB reverse loop primer: 5'-CCTTTAAGGCTTGTAACCCCA-3' (SEQ ID NO. 5).
3. The helicobacter pylori LAMP detection system according to claim 2, wherein the LAMP detection system comprises: 1 μ L of each of 5pM outer primers F3 and B3 in a 25 μ LLAMP reaction system; 1 mu L of each of 40pM of inner primers FIP and BIP; reverse loop primer LB of 20pM 1. mu.L; 2 × 12.5 μ L of reaction buffer; bst DNA polymerase 1 u L; fluorescent dye 1 μ L; DNA template 2. mu.L, the remainder was made up with sterile purified water.
4. A kit for rapidly detecting helicobacter pylori LAMP, which is characterized by comprising the primer group of claim 1 or the detection system of any one of claims 2 to 3.
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JP2010130931A (en) * 2008-12-03 2010-06-17 Toshiba Corp Primer set for specifically amplifying nucleic acid derived from bacterium belonging to genus helicobacter, and method for sensing and/or classifying the bacterium

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010130931A (en) * 2008-12-03 2010-06-17 Toshiba Corp Primer set for specifically amplifying nucleic acid derived from bacterium belonging to genus helicobacter, and method for sensing and/or classifying the bacterium

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* Cited by examiner, † Cited by third party
Title
Use of a Combination of Brushing Technique and the Loop-Mediated;Masaaki Minami et al.;《JOURNAL OF CLINICAL MICROBIOLOGY》;20061130;第44卷(第11期);第4032-4037页 *

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