CN114752692B - Primer probe combination for lactobacillus plantarum detection, RPA detection kit and detection method thereof - Google Patents
Primer probe combination for lactobacillus plantarum detection, RPA detection kit and detection method thereof Download PDFInfo
- Publication number
- CN114752692B CN114752692B CN202210316627.2A CN202210316627A CN114752692B CN 114752692 B CN114752692 B CN 114752692B CN 202210316627 A CN202210316627 A CN 202210316627A CN 114752692 B CN114752692 B CN 114752692B
- Authority
- CN
- China
- Prior art keywords
- detection
- lactobacillus plantarum
- primer
- nucleic acid
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 73
- 239000000523 sample Substances 0.000 title claims abstract description 55
- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 46
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 46
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 46
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 5
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 4
- 108020004707 nucleic acids Proteins 0.000 claims description 29
- 102000039446 nucleic acids Human genes 0.000 claims description 29
- 150000007523 nucleic acids Chemical class 0.000 claims description 29
- 238000006243 chemical reaction Methods 0.000 claims description 25
- 239000007853 buffer solution Substances 0.000 claims description 20
- 239000003795 chemical substances by application Substances 0.000 claims description 20
- 230000003321 amplification Effects 0.000 claims description 18
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 101710163270 Nuclease Proteins 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 239000008118 PEG 6000 Substances 0.000 claims description 6
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 6
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 102000052510 DNA-Binding Proteins Human genes 0.000 claims description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 102000016943 Muramidase Human genes 0.000 claims description 3
- 108010014251 Muramidase Proteins 0.000 claims description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- 102000018120 Recombinases Human genes 0.000 claims description 3
- 108010091086 Recombinases Proteins 0.000 claims description 3
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 3
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000006073 displacement reaction Methods 0.000 claims description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 3
- 229960000274 lysozyme Drugs 0.000 claims description 3
- 235000010335 lysozyme Nutrition 0.000 claims description 3
- 239000004325 lysozyme Substances 0.000 claims description 3
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 3
- 229940069446 magnesium acetate Drugs 0.000 claims description 3
- 235000011285 magnesium acetate Nutrition 0.000 claims description 3
- 239000011654 magnesium acetate Substances 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 3
- 230000000171 quenching effect Effects 0.000 claims description 3
- 101710096438 DNA-binding protein Proteins 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 239000007850 fluorescent dye Substances 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000006041 probiotic Substances 0.000 description 4
- 235000018291 probiotics Nutrition 0.000 description 4
- 241000186660 Lactobacillus Species 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 230000000529 probiotic effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 208000035657 Abasia Diseases 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 101710116602 DNA-Binding protein G5P Proteins 0.000 description 2
- 101710162453 Replication factor A Proteins 0.000 description 2
- 101710176758 Replication protein A 70 kDa DNA-binding subunit Proteins 0.000 description 2
- 101710176276 SSB protein Proteins 0.000 description 2
- 101710126859 Single-stranded DNA-binding protein Proteins 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- QSJXEFYPDANLFS-UHFFFAOYSA-N Diacetyl Chemical group CC(=O)C(C)=O QSJXEFYPDANLFS-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 1
- 201000010538 Lactose Intolerance Diseases 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003239 susceptibility assay Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a primer probe combination for lactobacillus plantarum detection, which is characterized in that the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of a gene probe is shown as SEQ ID NO. 3. The primer probe provided by the invention is designed and screened for a conserved region of a lactobacillus plantarum genome, and has good specificity and sensitivity and low detection limit.
Description
Technical Field
The invention belongs to the technical field of microorganism detection, and particularly relates to a primer probe combination, an RPA detection kit and a detection method for lactobacillus plantarum detection.
Background
Lactobacillus is gram positive, anaerobic or facultative anaerobic, bacillus-free and widely distributed in nature. Lactobacillus plantarum is one of lactobacillus, and compared with other lactobacillus, the lactobacillus plantarum has higher activity and can produce a large amount of acid. Scientific researches show that the lactobacillus plantarum has an inhibition effect on various pathogenic bacteria, inhibits the growth of the pathogenic bacteria by competing with the pathogenic bacteria for limiting nutrients, and also has the effect on other bacteria by the produced lactic acid, diacetyl and other substances, and adjusts the relationship among intestinal flora and the microecological composition. As a probiotic, lactobacillus plantarum also has various health care effects such as regulating immune function, promoting nutrient absorption, alleviating lactose intolerance, inhibiting tumor cell formation, and lowering serum cholesterol to prevent cardiovascular diseases, etc. In addition, lactobacillus plantarum is also widely applied to the fields of food fermentation, biological preservation and the like, and is a novel green additive. The related industries have wide application prospects.
At present, conventional probiotics detection is mainly carried out by biochemical reaction, selective culture counting and other methods, is not rapid and accurate, and limits development and application of probiotic products. With the rapid development of molecular biology techniques, nucleic acid detection methods based on sequence amplification are widely used. The recombinant enzyme polymerase amplification (RPA) technology has the advantages of low cost, high speed, high sensitivity and the like, and can reduce the detection cost, simplify the experimental process and improve the accuracy of results when being applied to lactobacillus plantarum detection, thereby helping the development of the probiotic industry.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a primer probe combination, an RPA detection kit and a detection method for lactobacillus plantarum detection, which have the advantages of strong specificity, high sensitivity, good repeatability, simplified detection steps and improved detection efficiency.
The invention provides the following technical scheme:
the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the gene probe is shown as SEQ ID NO. 3.
Further, the final concentration of the upper and lower primers applied to the PCR reaction was 0.4. Mu.M, and the final concentration of the fluorescent probe applied to the PCR reaction was 0.12. Mu.M.
Further, SEQ ID NO.3 shows that the 34 th base marks FAM fluorescent group from the 5' end of the fluorescent marking probe sequence, the 35 th base is an abasic site, and the 37 th base marks BHQ1 quenching group.
An application of primer probe combination in lactobacillus plantarum RPA detection kit.
A lactobacillus plantarum RPA detection kit adopting a primer probe combination comprises the primer probe combination, a rapid nucleic acid releasing agent, a buffer solution system A, a buffer solution system B and a dry powder reaction tube.
Further, the rapid nucleic acid releasing agent comprises lysozyme with the mass concentration of 1 mg/mL-20 mg/mL, polyethylene glycol with the volume percentage of 1% -5%, dithiothreitol with the molar concentration of 0.5 mM-5 mM and Tris-HCl with the molar concentration of 1 mM-10 mM.
Further, the buffer system A comprises PEG-6000 and water without nuclease, the buffer system B comprises magnesium acetate and water without nuclease, and the dry powder reaction tube comprises single-stranded DNA binding protein, recombinase, strand displacement DNA polymerase, trehalose, PEG-6000 and dNTPs.
A detection method using lactobacillus plantarum RPA detection kit comprises the following steps:
(1) Fully mixing a sample to be tested with a rapid nucleic acid releasing agent, wherein the mixing ratio of the nucleic acid releasing agent to the sample is 3: 3-20, incubating the mixture at 25-95 ℃ for 1-10 min to finish nucleic acid release;
(2) To each reaction tube were added in order 29.4. Mu.L of buffer system A, 2. Mu.L of upstream primer, 2. Mu.L of downstream primer, 0.6. Mu.L of fluorescent probe and 8.5. Mu.L of nuclease-free water;
(3) Adding 5 μl of a mixture of the sample and the nucleic acid releasing agent to each reaction tube;
(4) 2.5 mu L of buffer solution system B is added into each reaction tube, and after uniform mixing and centrifugation, the machine-on detection is carried out, and the reaction procedure is as follows: and (3) reacting for 20min at 42 ℃, reading a fluorescence value every 30s, and judging whether lactobacillus plantarum is detected.
Further, in the step (4), whether an exponential amplification curve is generated in the amplification process is firstly judged, and if the exponential amplification curve is not generated, the judgment is negative;
an exponential amplification curve was generated and interpreted according to the following table:
。
by adopting the technical scheme, the invention has the following beneficial effects:
1. the primer probe provided by the invention is designed and screened for a conserved region of a lactobacillus plantarum genome, and has good specificity and sensitivity and low detection limit.
2. The rapid nucleic acid releasing agent can complete the sample nucleic acid releasing process by simply mixing and incubating the rapid nucleic acid releasing agent with a sample, improves the extraction efficiency, reduces the dosage of reagents and samples, simplifies the operation steps, reduces the labor cost, and has the advantages of high efficiency, low cost, high quality and the like. The treated sample can be directly used for RPA detection without further purification.
3. The lactobacillus plantarum detection method combines the isothermal RPA detection technology with the rapid nucleic acid releasing agent, can complete the whole detection process from a sample to a result in about 30 minutes, and greatly improves the detection efficiency.
Drawings
FIG. 1 is a schematic diagram showing the results of a specific assay for lactobacillus plantarum detection using the kit of the present invention.
FIGS. 2-4 are schematic representations of three results of a sensitivity assay for lactobacillus plantarum detection using the kit of the present invention.
FIG. 5 is a schematic diagram showing the results of repeated experiments for lactobacillus plantarum detection using the kit of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the drawings and detailed description are only intended to illustrate the invention and are not intended to limit the invention.
Example 1
The invention provides a primer probe combination for lactobacillus plantarum detection, which selects a conserved specific sequence as a detection target through BLAST comparison according to a lactobacillus plantarum whole genome sequence provided by NCBI database, and the sequence is as follows as shown in SEQ ID No. 4:
5’-ATGCCATTTAATGGTTATCAGACGTATTATCGAATCGTAGGGGATCGGCAATCAAATAAGACACCGTTAGTTTTGCTACATGGTGGTCCAGGATCAACGCATAATTACTTTGAAGGATTTGATGACCTGGCCGTTCAAACGGGACGACCAATCGTCATGTATGATCAATTAGGCTGTGGGCGTTCATCGATACCTGATGACGATCAATTATGGCAAGCCGCAACGTGGGTGGCAGAGTTACAGGCGTTACGTACGTATTTAGACTTACCCGAGATTCACTTACTAGGGCAGTCCTGGGGTGGTATGCTAGCTATTATTTATGGTTGTGACTATCGACCACAGGGGATTAAAAGTTTGATTTTAGCCAGTACCTTGTCTTCAGCCCGACTGTGGGCTCAGGAACAGCATCGGATGATTCGGTTAATGTCCCCAGCGGATTAGTCGGCTATTGCGACAGCAGAACGGTTACAGGATTTCACAGGTGCTGCCTACCTGACTGCTAATCAACATTTTATGACGCAACATGCGTCGGGCCCGATTACTGCTGATGATCCTGAATTCCTACGGCGATCAAAACGGGTGGGGACCACGGCTTATAATGTAGCGTGGGGTCCCAATGAATATAATCCGACTGGTACGTTGGCGGATTATGAGTATACCGACCGATTACAGTATTTGCAGATGCCAACTTTAGTCACCAGTGGAACGGATGATTTGTGTACGCCATTAGTCGCTAAGACCATGGTCGACCAGTTACCTCATGCGACTTGGACGTTATTTCCCCGTAGCCGTCACATGGCGTTTGTCGATGAAAACACGGCCTATATGACGCGATTGCGTCACTGGCTAGCAGCCCATGATTAG-3’
according to the requirements of the detection target sequence and the primer probe, a primer probe combination for lactobacillus plantarum specificity detection is designed and obtained, and the primer probe combination comprises an upstream primer, a downstream primer and a fluorescent probe. The nucleotide sequences of the upstream primer and the downstream primer are shown as SEQ ID No.1 and SEQ ID No.2, and the nucleotide sequence of the fluorescent probe is shown as SEQ ID No.3, and the nucleotide sequences are as follows:
SEQ ID NO.1：
5’-TCCCAATGAATATAATCCGACTGGTACGTTG-3’。
SEQ ID NO.2：
5’-TAACTGGTCGACCATGGTCTTAGCGACTAATG-3’
SEQ ID NO.3：5’-CCGACCGATTACAGTATTTGCAGATGCCAACTTGCACCAGTGGAACGGA-3’
the final concentration of each primer applied to the PCR reaction was 0.4. Mu.M, and the final concentration of the gene probe applied to the PCR reaction was 0.12. Mu.M. SEQ ID NO.3 shows that the 34 th base marks FAM fluorescent group from the 5' end of the fluorescent marked probe sequence, the 35 th base is an abasic site, and the 37 th base marks BHQ1 quenching group.
Example 2
The primer probe combination can be applied to a lactobacillus plantarum RPA detection kit.
The invention provides a lactobacillus plantarum RPA detection kit adopting primer probe combination.
The kit also comprises a rapid nucleic acid releasing agent, a buffer solution system A, a buffer solution system B and a dry powder reaction tube.
The rapid nucleic acid releasing agent comprises lysozyme with the mass concentration of 2.5mg/mL, polyethylene glycol with the volume percentage of 2.5%, dithiothreitol with the molar concentration of 1mM and Tris-HCl with the molar concentration of 5 mM.
Wherein the buffer solution system A comprises PEG-6000 and water without nuclease, the buffer solution system B comprises magnesium acetate and water without nuclease, and the dry powder reaction tube comprises single-stranded DNA binding protein, recombinase, strand displacement DNA polymerase, trehalose, PEG-6000 and dNTPs.
In this example, buffer system A, buffer system B and dry powder reaction tubes were purchased from Weifang Anpu future Biotechnology Co.
The embodiment provides a method for detecting lactobacillus plantarum by using the extraction-free RPA isothermal rapid detection kit, which comprises the following steps:
(1) Fully mixing a sample to be tested with a rapid nucleic acid releasing agent, wherein the mixing ratio of the nucleic acid releasing agent to the sample is 3: 3-20, wherein the optimal ratio is 1:1, and incubating the mixture at 25-95 ℃ and the optimal temperature is 37 ℃ for 1-10 min to finish the release of nucleic acid;
(2) To each reaction tube were added in order 29.4. Mu.L of buffer system A, 2. Mu.L of upstream primer, 2. Mu.L of downstream primer, 0.6. Mu.L of fluorescent probe and 8.5. Mu.L of nuclease-free water;
(3) Adding 5 μl of a mixture of the sample and the nucleic acid releasing agent to each reaction tube;
(4) 2.5 mu L of buffer solution system B is added into each reaction tube, and after uniform mixing and centrifugation, the machine-on detection is carried out, and the reaction procedure is as follows: the reaction was carried out at 42℃for 20min, and the fluorescence value was read every 30 s.
In the step (4), whether an exponential amplification curve is generated in the amplification process is firstly judged, and the situation that the exponential amplification curve is not generated is judged as negative;
an exponential amplification curve was generated and interpreted according to table 1:
table 1 principle of interpretation of test results
The Ct value represents the number of cycles when the amplification curve crosses the set threshold line, and the smaller the Ct value, the fewer PCR cycles are required when the fluorescence intensity reaches the threshold. The higher the concentration of target sequences in the sample; the larger the Ct value, the more PCR cycles are required when the fluorescence intensity reaches the threshold. The lower the concentration of target sequence in the sample.
Example 3
Specific assay for lactobacillus plantarum detection using the kit of the invention
The RPA test was performed using lactobacillus plantarum (ATCC 8014), lactobacillus rhamnosus (ATCC 53103), staphylococcus aureus (ATCC 29213) and escherichia coli (ATCC 43889) as samples according to the test method described in example 2, and 3 replicates were performed for each strain. To evaluate the specificity of the combination of the kit and the detection method. As shown in FIG. 1, only 3 lactobacillus plantarum samples generate amplification curves, which shows that the primer probe provided by the invention has higher specificity and can accurately detect lactobacillus plantarum.
Example 4
The kit of the invention is used for measuring the sensitivity of lactobacillus plantarum detection.
The detection method described in example 2 was selectedSelecting concentration to be 10 5 、10 4 、10 3 、10 2 cfu/mL of lactobacillus plantarum liquid is taken as a sample, RPA detection is carried out, and 3 repeated experiments are carried out on each concentration gradient so as to evaluate the detection sensitivity of the combination of the kit and the detection method. Of which 10 5 、10 4 、10 3 The cfu/mL sample detection results are shown in FIGS. 2-4, 10 2 No amplification curve was seen for cfu/mL. Therefore, the detection limit of the method is 10 3 cfu/mL。
And (3) extracting nucleic acid from the gradient diluted lactobacillus plantarum liquid sample by using a commercial magnetic bead method bacterial DNA nucleic acid extraction and purification kit, detecting by using the primer probe and the RPA reaction system provided by the invention, and performing a control experiment. The detection conditions are shown in Table 2. It can be seen that the lowest concentration of 10 can be detected in both example 4 and the comparative example 3 cfu/mL of lactobacillus plantarum, and the cfu/mL of lactobacillus plantarum and the lactobacillus plantarum have higher consistency. The rapid nucleic acid releasing agent provided by the invention can effectively lyse bacteria, has higher nucleic acid releasing efficiency, and the sample treated by the rapid nucleic acid releasing agent can realize RPA detection sensitivity equivalent to that of the sample treated by a commercial magnetic bead method extraction kit.
TABLE 2 sensitivity measurement experiment results
Wherein "+" indicates positive detection result, and "-" indicates negative detection result
Table 3 shows the time-consuming comparison of the detection method of the present invention and the conventional bacterial nucleic acid detection method, and shows that the detection method of the present invention shortens the time-consuming two steps of nucleic acid extraction and amplification detection, respectively, and has the characteristics of good efficiency, low cost and high sensitivity
Table 3 comparison of time consuming assay
Example 5
The lactobacillus plantarum RPA rapid detection kit and the repeatability evaluation of the detection method are used.
For 10, the detection method described in example 2 was followed 5 And 10 4 The recombinant plasmid standard containing the lactobacillus plantarum target sequence was tested at each concentration for 4 replicates for evaluating the reproducibility of the test of the kit and test method combination. The results are shown in FIG. 5, and Ct values are shown in Table 4, and the results show that the method has better repeatability.
TABLE 4 repeatability evaluation
The embodiment shows that the combination of the kit and the detection method provided by the invention has the advantages of strong specificity, high sensitivity and good repeatability, and can accurately detect the lactobacillus plantarum in the sample within about 30 minutes.
The foregoing examples merely illustrate embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> national institute of inspection and quarantine science
<120> primer probe combination for lactobacillus plantarum detection, RPA detection kit and detection method thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> DNA
<213> SEQ ID NO.1 (Artificial sequence)
<400> 1
tcccaatgaa tataatccga ctggtacgtt g 31
<210> 2
<211> 32
<212> DNA
<213> SEQ ID NO.2 (Artificial sequence)
<400> 2
taactggtcg accatggtct tagcgactaa tg 32
<210> 3
<211> 49
<212> DNA
<213> SEQ ID NO.3 (Artificial sequence)
<400> 3
ccgaccgatt acagtatttg cagatgccaa cttgcaccag tggaacgga 49
<210> 4
<211> 864
<212> DNA
<213> SEQ ID No. 4 (Artificial sequence)
<400> 4
atgccattta atggttatca gacgtattat cgaatcgtag gggatcggca atcaaataag 60
acaccgttag ttttgctaca tggtggtcca ggatcaacgc ataattactt tgaaggattt 120
gatgacctgg ccgttcaaac gggacgacca atcgtcatgt atgatcaatt aggctgtggg 180
cgttcatcga tacctgatga cgatcaatta tggcaagccg caacgtgggt ggcagagtta 240
caggcgttac gtacgtattt agacttaccc gagattcact tactagggca gtcctggggt 300
ggtatgctag ctattattta tggttgtgac tatcgaccac aggggattaa aagtttgatt 360
ttagccagta ccttgtcttc agcccgactg tgggctcagg aacagcatcg gatgattcgg 420
ttaatgtccc cagcggatta gtcggctatt gcgacagcag aacggttaca ggatttcaca 480
ggtgctgcct acctgactgc taatcaacat tttatgacgc aacatgcgtc gggcccgatt 540
actgctgatg atcctgaatt cctacggcga tcaaaacggg tggggaccac ggcttataat 600
gtagcgtggg gtcccaatga atataatccg actggtacgt tggcggatta tgagtatacc 660
gaccgattac agtatttgca gatgccaact ttagtcacca gtggaacgga tgatttgtgt 720
acgccattag tcgctaagac catggtcgac cagttacctc atgcgacttg gacgttattt 780
ccccgtagcc gtcacatggc gtttgtcgat gaaaacacgg cctatatgac gcgattgcgt 840
cactggctag cagcccatga ttag 864
Claims (7)
1. A primer probe combination for lactobacillus plantarum detection is characterized in that the nucleotide sequences of an upstream primer and a downstream primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
2. The primer probe combination of claim 1, wherein the final concentration of the upstream primer and the downstream primer is 0.4. Mu.M, and the final concentration of the probe is 0.12. Mu.M.
3. The primer probe combination of claim 1, wherein the 5' end of the probe sequence is provided with a 34 th base labeled FAM fluorescent group, a 35 th base site is provided with a 37 th base labeled BHQ1 quenching group.
4. Use of the primer probe combination of claim 1 in a lactobacillus plantarum RPA detection kit.
5. A lactobacillus plantarum RPA detection kit adopting the primer probe combination of claim 1, which is characterized by comprising the primer probe combination of claim 1, a rapid nucleic acid releasing agent, a buffer solution system a, a buffer solution system B and a dry powder reaction tube; the rapid nucleic acid releasing agent comprises lysozyme with the mass concentration of 1-20 mg/mL, polyethylene glycol with the volume percentage of 1-5%, dithiothreitol with the molar concentration of 0.5-5 mM and Tris-HCl with the molar concentration of 1-10 mM; the buffer solution system A comprises PEG-6000 and water without nuclease, the buffer solution system B comprises magnesium acetate and water without nuclease, and the dry powder reaction tube comprises single-chain DNA binding protein, recombinase, strand displacement DNA polymerase, trehalose, PEG-6000 and dNTPs.
6. A detection method using the lactobacillus plantarum RPA detection kit of claim 5, comprising the steps of:
(1) Fully mixing a sample to be tested with a rapid nucleic acid releasing agent, wherein the mixing ratio of the nucleic acid releasing agent to the sample is 3: 3-20, incubating the mixture at 25-95 ℃ for 1-10 min to finish nucleic acid release;
(2) To each reaction tube were added in order 29.4. Mu.L of buffer system A, 2. Mu.L of upstream primer, 2. Mu.L of downstream primer, 0.6. Mu.L of probe and 8.5. Mu.L of nuclease free water;
(3) Adding 5 μl of a mixture of the sample and the nucleic acid releasing agent to each reaction tube;
(4) 2.5 mu L of buffer solution system B is added into each reaction tube, and after uniform mixing and centrifugation, the machine-on detection is carried out, and the reaction procedure is as follows: and (3) reacting for 20min at 42 ℃, reading a fluorescence value every 30s, and judging whether lactobacillus plantarum is detected.
7. The method according to claim 6, wherein in the step (4), it is determined whether an exponential amplification curve is generated during the amplification process, and it is determined that the exponential amplification curve is not generated;
an exponential amplification curve was generated and interpreted according to the following table:
。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210316627.2A CN114752692B (en) | 2022-03-29 | 2022-03-29 | Primer probe combination for lactobacillus plantarum detection, RPA detection kit and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210316627.2A CN114752692B (en) | 2022-03-29 | 2022-03-29 | Primer probe combination for lactobacillus plantarum detection, RPA detection kit and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114752692A CN114752692A (en) | 2022-07-15 |
| CN114752692B true CN114752692B (en) | 2024-03-19 |
Family
ID=82326633
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210316627.2A Active CN114752692B (en) | 2022-03-29 | 2022-03-29 | Primer probe combination for lactobacillus plantarum detection, RPA detection kit and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114752692B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107981360A (en) * | 2017-11-24 | 2018-05-04 | 华东理工大学 | A kind of application of lactobacillus plantarum X7021 |
| CN108441542A (en) * | 2018-04-17 | 2018-08-24 | 广州市妇女儿童医疗中心 | Streptococcusagalactiae visualization kit for detecting nucleic acid based on recombinase polymeric enzymatic amplification technology and method |
| CN110423827A (en) * | 2018-05-23 | 2019-11-08 | 海南大学 | A kind of fast inspection technology in loop-mediated isothermal amplification of streptococcus agalactiae scene based on pcsB gene |
-
2022
- 2022-03-29 CN CN202210316627.2A patent/CN114752692B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107981360A (en) * | 2017-11-24 | 2018-05-04 | 华东理工大学 | A kind of application of lactobacillus plantarum X7021 |
| CN108441542A (en) * | 2018-04-17 | 2018-08-24 | 广州市妇女儿童医疗中心 | Streptococcusagalactiae visualization kit for detecting nucleic acid based on recombinase polymeric enzymatic amplification technology and method |
| CN110423827A (en) * | 2018-05-23 | 2019-11-08 | 海南大学 | A kind of fast inspection technology in loop-mediated isothermal amplification of streptococcus agalactiae scene based on pcsB gene |
Non-Patent Citations (6)
| Title |
|---|
| Dissecting of the AI-2/LuxS Mediated Growth Characteristics and Bacteriostatic Ability of Lactiplantibacillus plantarum SS-128 by Integration of Transcriptomics and Metabolomics;Yilin Qian 等;《Foods》;20220222;第11卷(第638期);第1-18页 * |
| Extraction, purification and characterization of low molecular weightProline iminopeptidase from probiotic L. plantarum for meattenderization;Preeti Chanalia 等;《International Journal of Biological Macromolecules》;20171221;第109卷;第651-663页 * |
| Muhammad,S. 等.CP024413.1 Lactiplantibacillus plantarum strain ATCC 8014 chromosome, complete genome.《GenBank》.2017,第1-2页. * |
| Rapid PCR-based procedure to identify lactic acid bacteria: application to six common Lactobacillus species;Patrice Chagnaud 等;《Journal of Microbiological Methods》;20211231;第44卷;第139-148页 * |
| Recombinase polymerase amplification: Basics, applications and recent advances;Ivan Magrina Lobato 等;《Trends in Analytical Chemistry》;20171026;第98卷;第19-35页 * |
| 益生菌快速检测技术研究进展;吕秀莉 等;《食品与机械》;20211130;第37卷(第11期);第198-204、220页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114752692A (en) | 2022-07-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2596059C (en) | Method of quantitatively analysing microorganism targeting rrna | |
| CN110734988B (en) | Constant-temperature amplification method for methicillin-resistant staphylococcus aureus (MRSA) nucleic acid | |
| CN114752692B (en) | Primer probe combination for lactobacillus plantarum detection, RPA detection kit and detection method thereof | |
| CN116814821A (en) | Primer probe combination, kit and application for detecting 4 living bacteria in micro-ecological four-linked living bacteria product | |
| CN115976238B (en) | qPCR rapid detection kit containing 15 mycoplasma, and use method and application thereof | |
| CN108192988B (en) | Staphylococcus aureus strand exchange amplification detection method | |
| JP2010081889A (en) | Pcr primer for detecting lactic bacterium | |
| Pang et al. | Cyclic strand displacement polymerase reaction to turn-on molecular beacons for rapid detection of Staphylococcus aureus | |
| CN111378724B (en) | RNA amplification detection method and detection kit | |
| CN106148483A (en) | The primer of detection Bacillus coli cells DNA and method | |
| CN111849966A (en) | Constant temperature detection method for identifying lactobacillus brevis and special primer and kit thereof | |
| CN111996271A (en) | RPA detection primer group, kit and method for drug-resistant gene NDM-1 of superbacteria | |
| CN108504754A (en) | Green Wei Si Salmonellas real-time fluorescence quantitative PCR quick detection kit and its detection method | |
| KR102655121B1 (en) | A Primer set for specifically detecting Lactobacillus sakei K040706 and uses thereof | |
| CN113355330B (en) | ssDNA aptamer for specifically recognizing Weissella viridescens and screening method and application thereof | |
| CN110512017B (en) | Mouse corynebacterium fluorescent quantitative PCR detection kit and primer | |
| US20230242999A1 (en) | Quantitative detection method for six antibiotic drug resistant genes in aquatic product | |
| CN116121272A (en) | Nucleic acid detection target and method for lactobacillus salivarius | |
| CN114854883A (en) | Special primer and kit for identifying pediococcus pentosaceus | |
| CN118028505A (en) | Dual PCR primer group for simultaneously detecting two probiotics and method and kit thereof | |
| CN113621720A (en) | Multiple fluorescent quantitative PCR method for detecting and identifying 3 food-borne pathogenic bacteria | |
| CN116590446A (en) | Specific primer for lactobacillus equi-producing lactobacillus equi subspecies, detection method and application | |
| CN116479147A (en) | Primer probe and kit for detecting burkholderia meliotis and application of primer probe and kit | |
| CN116904628A (en) | Primer combination and kit for detecting three pathogenic bacteria of pandas and detection method of primer combination and kit | |
| CN117947194A (en) | Indiana salmonella molecular detection method and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |