CN117448467A - Two-set primer probe combination and method for rapidly detecting and identifying lactobacillus rhamnosus HN001 at strain level - Google Patents
Two-set primer probe combination and method for rapidly detecting and identifying lactobacillus rhamnosus HN001 at strain level Download PDFInfo
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Abstract
The invention relates to two groups of specific primers and probes of lactobacillus rhamnosus HN001, and the two groups of primers and probes can rapidly and accurately identify lactobacillus rhamnosus HN001 at the strain level. The invention also relates to a qPCR detection method for determining lactobacillus rhamnosus HN001, the method comprising the use of specific oligonucleotide primers and probes for lactobacillus rhamnosus HN001. The invention also relates to a qPCR detection kit for lactobacillus rhamnosus HN001, which comprises a specific oligonucleotide primer and a probe. The method utilizes genome comparison to find out the specific gene of the lactobacillus rhamnosus HN001, and realizes the identification of the lactobacillus rhamnosus HN001 at the strain level by designing a primer through the specific gene. The comparison result of the genome finds the RepA gene and the ferritin-like domain-containing gene on the lactobacillus rhamnosus HN001, the specificity of the recombinant lactobacillus rhamnosus is confirmed through sequence similarity comparison, and then specific primers and probes are designed. According to the method, the lactobacillus rhamnosus HN001 in food or health-care food can be identified through qPCR, and whether a sample to be detected is the lactobacillus rhamnosus HN001 is judged through observing an amplification curve and a Ct value in a qPCR result.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a high-specificity and high-sensitivity primer pair and a high-sensitivity probe based on a qPCR technology, a qPCR detection kit containing the primer pair and the probe, a qPCR detection method for detecting lactobacillus rhamnosus HN001, and application of the primer pair and the probe or the kit in rapid detection of lactobacillus rhamnosus HN001. The invention can be used for rapidly detecting and identifying lactobacillus rhamnosus HN001 at the strain level, and can be applied to detection laboratories, detection institutions and the like.
Background
Lactobacillus rhamnosus HN001 is one of the most well studied probiotic strains, is very common in commercial probiotic products, and has been derived from healthy human bodies, and lactobacillus rhamnosus HN001 is already a member of the "list of strains available for infant food" published by the national ministry of health, and has been widely used in dairy products and infant food at present. Lactobacillus rhamnosus HN001 has acid resistance, bile salt resistance, strong adhesiveness and constant value capability in human bodies, and effective biological activity for infants, and has clear and long-term clinical evidence to prove that lactobacillus rhamnosus HN001 can support the development of the infant immune system, can obviously reduce the prevalence of eczema and inhibit anaphylactic reaction compared with a placebo group, is beneficial to intestinal health, and relieves the constipation and diarrhea symptoms of infants.
In view of the versatility of lactobacillus rhamnosus HN001, many milk products, infant food and health food have been added with the bacterium. The function of the probiotics has the specificity of the strains, and the accurate identification of the probiotics at the strain level is an important basis for evaluating the safety evaluation and the functionality of the probiotics. However, lactobacillus rhamnosus has a large variety and similar phenotype and physicochemical properties, and is difficult to rapidly identify and distinguish at the strain level. The traditional identification method mainly comprises biochemical identification, and has the advantages of complicated steps, long inspection period, poor distinguishing capability and difficulty in rapidly and accurately identifying lactobacillus rhamnosus HN001. It is therefore desirable to establish a method that is capable of rapidly and accurately identifying lactobacillus rhamnosus HN001 at the strain level, in order to meet the accuracy and timeliness of the identification of probiotic products with lactobacillus rhamnosus HN001 as an effective strain.
At present, gene analysis technologies such as Whole Genome Sequencing (WGS), 16SrRNA sequencing, multi-point sequence analysis (MLST), pulsed Field Gel Electrophoresis (PFGE) and CRISPR are generally adopted to accurately identify strains, and these identification methods mainly depend on gene identification, however, the gene identification technologies are often time-consuming, require expert skills or advanced hardware and software, and are difficult to meet frequent and massive identification requirements. The strain-specific PCR method can be performed directly without advanced instrumentation, and only the strain-specific sequences need to be identified. Genomic comparison analysis can search for strain-specific genes, helping to identify strain-specific gene sequences. Through genome alignment, a plurality of strain-specific sequences can be identified, and a plurality of strain-specific primer pairs can be designed more reasonably and controllably. The series connection of a plurality of strain-specific primer pairs enables the identification result to be more reliable. The strain-specific PCR identification method is accurate, quick, economical, good in repeatability and simple and convenient to operate, and can meet the requirement of frequent detection of products containing lactobacillus rhamnosus HN001.
The probe method qPCR is to add a fluorescent probe (hydrolysis probe) into a PCR system, and the distance between two groups on the probe is changed by utilizing the 5' end exonuclease function of polymerase in the PCR reaction through a fluorescence resonance energy transfer principle, so that the fluorescence intensity or the fluorescence type in the system is changed, and the change is directly related to the type and the quantity of a PCR product, thereby achieving the purposes of quantification and identification. The qPCR detection technology of the qPCR probe method has the advantages of strong specificity, high detection speed, high sensitivity, good repeatability and the like. Compared with the common PCR amplification, the method omits the step of gel electrophoresis, and can identify whether the sample contains lactobacillus rhamnosus HN001 by only observing whether a specific amplification curve exists. Zhou Liguang et al used qPCR to establish a method for identifying bifidobacteria and lactobacillus fermentum in a composite probiotic product, hu Huilong et al established a method for identifying lactobacillus fermentum based on qPCR technology. The qPCR technology is applied to the field of detection and identification of probiotics more and more, and plays an increasingly important role in the field of detection and identification of probiotics. In order to improve the specificity of the primers, the inventor finds out that two strain-level specific genes of lactobacillus rhamnosus HN001 are a RepA gene and a ferritin-like domain-rotation gene respectively through genome comparison, and designs the primers by taking the two genes as target sequences, and establishes a method for rapidly and accurately identifying lactobacillus rhamnosus HN001 at the strain level through repeated experiments and groping. The method is suitable for detection and identification of lactobacillus rhamnosus HN001.
Disclosure of Invention
It is an object of the present invention to provide qPCR specific primers and probes that can rapidly and accurately identify and differentiate lactobacillus rhamnosus HN001 at the strain level.
It is another object of the present invention to provide a method for identifying lactobacillus rhamnosus HN001 by qPCR specific amplification using the two sets of primer probes.
In order to achieve the above object, the present invention is achieved by the following technical scheme.
In one embodiment, the genome file of 11 groups of Lactobacillus rhamnosus HN001 homologous kindred strains is downloaded for genome alignment, comprising the following steps.
(1) Genome files of 11 different lactobacillus rhamnosus including lactobacillus rhamnosus HN001 are downloaded through NCBI, specific proteins only existing in lactobacillus rhamnosus HN001 are found out through comparing the proteome files, and then target gene sequences are obtained according to corresponding gene accession numbers of the specific proteins in the annotation files. The specific genes found in the genome alignment are RepA gene and ferritin-like domain-containing gene respectively, the corresponding NCBI accession numbers of the genes are NC_011223.1 sequence starting positions 8203-8754 and NZ_ABWJ01000050.1 sequence starting positions 36-584 respectively.
(2) After the sequence of the specific gene is obtained, in order to ensure the specificity of the specific gene in lactobacillus rhamnosus HN001, the sequence similarity alignment is carried out on the target sequence through the BLAST function of NCBI, the BLAST result is good, and only lactobacillus rhamnosus HN001 has the highest similarity with the target sequence. And then designing specific primers and probes, carrying out multiple rounds of screening on the primers after the design, and preliminarily determining the specificity of the primers and the probes on NCBI through primBLAST to ensure that the amplified products of the primers and the probes are only lactobacillus rhamnosus HN001.
(3) Primer specificity verification: to ensure the specificity of the strain level of the designed primers and probes, the present inventors used 23 strains closely related to lactobacillus rhamnosus HN001 as non-target strains, which contained 3 different sources of lactobacillus rhamnosus HN001, lactobacillus rhamnosus LGG, mp108, CTCC6001, ATCC7469, ATCC8530, ATCC11443 and 11 other different species of lactic acid bacteria as non-target strains.
In one embodiment, the two pairs of primers and probe sequences used for detecting and identifying lactobacillus rhamnosus HN001 of the invention are:
upstream primer Rp (F): 5'-GCTTAGCTTTCTCTACTGGCGA-3'
The downstream primer Rp (R): 5'-GTTTGCACCAGATCCCCTCA-3'
Probe Rp (P): 5'-AAGCGCCAAGCCTTACGTGAT-3'
Upstream primer FD (F): 5'-CATCCAAGCCTTCTCGTGGT-3'
Downstream primer FD (R): 5'-ACAACATTTGGTTGGCCTGCF-3'
Probe FD (P): 5'-GCAAGGCCTGCAGAGTAGCGA-3'.
In one embodiment, the probe is a Taqman probe.
In one embodiment, a fluorescence quenching group is attached to the 3 'end of the probe and a fluorescence reporting group is attached to the 5' end.
The fluorescence quenching group in the present invention may be a fluorescence quenching group commonly used in the art, such as BHQ3, BHQ1, BHQ2, TAMRA, or the like; the fluorescent reporter group may be a fluorescent reporter group commonly used in the art, such as FAM, HEX, VIC, or the like.
In one embodiment, the probe has a FAM attached to the 5 'end and a BHQ1 attached to the 3' end.
In one embodiment, the probe has a VIC attached to the 5 'end and BHQ1 attached to the 3' end.
In one aspect, the invention provides a kit for detecting lactobacillus rhamnosus HN001 by a qPCR method, wherein the kit comprises the primer pair and the probe of the invention.
In another aspect, the invention provides a method for detecting lactobacillus rhamnosus HN001 by a qPCR method, the method comprising using the primer pair and probe of the invention or the kit of the invention.
In one embodiment, the qPCR method for identifying lactobacillus rhamnosus HN001 is:
PCR reaction system: taqman Gene Expression MasterMix 12.5.5 μL; ddH 2 O8 μl; 1. Mu.L of the upstream primer (10. Mu. Mol/L); 1. Mu.L of a downstream primer (10. Mu. Mol/L);
0.5. Mu.L of probe (10. Mu. Mol/L); template DNA 2. Mu.L. qPCR amplification parameters: preheating at 95deg.C for 10min, and circulating at 95deg.C 15S and 58 deg.C 35S for 40 cycles.
Drawings
FIG. 1 is a specific assay of Rp primer probe set, in which 1,2,3 are three different sources of Lactobacillus rhamnosus HN001, blank and negative control, respectively: blank control was ddH 2 O, negative control was a closely related strain other than lactobacillus rhamnosus HN001.
FIG. 2 shows the specificity test of FD primer probe sets, wherein 1,2 and 3 are respectively three different sources of lactobacillus rhamnosus HN001, blank and negative control: blank control was ddH 2 O, negative control was a closely related strain other than lactobacillus rhamnosus HN001.
FIG. 3 is a sensitivity experiment of Rp primer probe set, and the result shows that the detection sensitivity of the qPCR method is that the template concentration is 20×10 -5 μg/mL。
FIG. 4 is a sensitivity experiment of FD primer probe set, and the result shows that the detection sensitivity of the qPCR method is 2×10 template concentration -3 μg/mL。
Table 1 shows the names and sources of the strains used for primer specificity verification.
Description of the embodiments
The present invention is further illustrated by way of examples, but the present invention is not limited to the following examples.
Example 1
(1) Materials and reagents
Test bacteria: the strains listed in Table 1 below are test bacteria used in this experiment.
The main reagent comprises: MRS medium, BD company, USA; phosphate buffered saline (phosphate buffered saline, PBS), beijing solibao technologies limited; plasmid DNA small extraction kit, bacterial genome DNA extraction kit, proteinase K (20 mg/mL), lysozyme (50 mg/mL), beijing TIANGEN company; taqman Gene Expression MasterMix, sieimer, USA; both primers and probes were synthesized by Shanghai Biotechnology Co., ltd.
TABLE 1 list of strains used in Lactobacillus rhamnosus HN001 primer specificity experiments
| Species of type | Strain name | Bacterial strain origin |
| Lactobacillus rhamnosus | HN001 | Milk powder sample separation |
| Lactobacillus rhamnosus | HN001 | Bacterial liquid sample separation |
| Lactobacillus rhamnosus | HN001 | DuPont of U.S |
| Lactobacillus rhamnosus | CICC6135 | China industry culture Collection center |
| Lactobacillus rhamnosus | Mp108 | Separation of bacterial powder sample |
| Lactobacillus rhamnosus | GG | Separation of bacterial powder sample |
| Lactobacillus rhamnosus | CICC6151 | China industry culture Collection center |
| Lactobacillus rhamnosus | ATCC6001 | China inspection and quarantine science institute center for culture collection |
| Lactobacillus casei | ATCC393 | China inspection and quarantine science institute center for culture collection |
| Lactobacillus casei | ATCC334 | China inspection and quarantine science institute center for culture collection |
| Lactobacillus plantarum | CTCC6009 | China inspection and quarantine science institute center for culture collection |
| Lactobacillus plantarum | ATCC8014 | China inspection and quarantine science institute center for culture collection |
| Lactobacillus delbrueckii | CICC6047 | China inspection and quarantine science institute center for culture collection |
| Lactobacillus casei | ATCC393 | China inspection and quarantine science institute center for culture collection |
| Lactobacillus reuteri acidophilus Lactobacillus acidophilus Fermented lactobacillus Swiss milk Bacillus brevis and bifidobacterium Bifidobacterium bifidum Bacillus bifidus | CTCC6226 CTCC6074ATCC4356 ATCC9338CICC603 2ATCC15700ATCC11 863CICC6071 | Chinese inspection and quarantine of China center for culture Collection of China inspection and quarantine science institute Chinese inspection and quarantine scientific institute bacteria of the national institute of sciences (China) culture Collection In the seed collection center of China inspection and quarantine science institute Chinese inspection and quarantine department of national inspection and quarantine science institute of research and culture collection Chinese inspection and quarantine scientific institute strain of academy of sciences China center for inspection and quarantine science institute of preservation (China center for type culture Collection) |
| Lactobacillus casei | ATCC334 | China inspection and quarantine science institute center for culture collection |
(2) Instrument and apparatus:
7500FAST real-time fluorescence PCR instrument, ABI company, USA; ultra-micro spectrophotometer NanoDrop One, sammer, usa; a thermostat water bath WNB14, membert, germany; biological safety cabinet class II class A2 NU-602, U.S. Nuaine; high speed centrifuges Sigama 1-16, sigama, germany; constant temperature incubator, binder Germany.
(3) Designing and synthesizing a primer: the specific genes RepA and ferritin-like domain-containing in lactobacillus rhamnosus HN001 are obtained through genome comparison, primer design is carried out according to the two specific gene sequences, the designed primers are firstly screened, primerBLAST is carried out on the screened primers on NCBI, the specificity of the primers is ensured, and through specificity experiment verification, two groups of primer sequences are finally determined to be shown as follows, and the primers are synthesized by Shanghai biological engineering technical service Co;
upstream primer Rp (F): 5'-GCTTAGCTTTCTCTACTGGCGA-3'
The downstream primer Rp (R): 5'-GTTTGCACCAGATCCCCTCA-3'
Probe Rp (P): 5'-FAM-AAGCGCCAAGCCTTACGTGAT-BHQ1-3'
Upstream primer FD (F): 5'-CATCCAAGCCTTCTCGTGGT-3'
Downstream primer FD (R): 5'-ACAACATTTGGTTGGCCTGCF-3'
Probe FD (P): 5'-VIC-GCAAGGCCTGCAGAGTAGCGA-BHQ1-3'.
(4) DNA extraction: placing the activated strain culture stock solution 1 mL of the 3 rd generation in a centrifugal machine for centrifugation at 12,000 r/min for 5 min by adopting a bacterial genome DNA extraction kit; pouring out the supernatant, wherein lactobacillus rhamnosus HN001 as gram-positive bacteria is added with lysozyme 70 mu L (50 mg/ml) and buffer solution TE 110 mu L, and digested at 37 ℃ for more than 0.5 h; and adding 20 mu L of proteinase K, and digesting for 10min at 70 ℃ in a buffer solution GB220 mu L provided in the bacterial genome DNA extraction kit. Adding 220 mu L of absolute ethyl alcohol, fully oscillating 15 and s, adding the oscillated solution into an adsorption column, centrifuging at 12000 rpm for 30 and s, and discarding waste liquid. 500 micro L buffer GD is added into the adsorption column after centrifugation, 30 s is centrifuged at 12000 rpm, and waste liquid is discarded. A rinse solution PW 600 μl was added to the column, centrifuged at 12000 rpm for 30 min s, the waste liquid was discarded, and the rinsing step was repeated twice. After rinsing, the solution was centrifuged at 12000 rpm for 2 min, the waste solution was discarded, and the column was left at room temperature for several minutes to air-dry the residual rinse solution. Transferring the adsorption column into a clean centrifuge tube, suspending and dripping 50-200 mu L of eluent TE into the middle part of the adsorption film, standing at room temperature for 2-5 min, centrifuging at 12000 rpm for 2 min, collecting the solution into the centrifuge tube, and preserving at-20deg.C.
(5) PCR reaction system and PCR reaction procedure: reagents and DNA solution were added to a 0.1 ml PCR tube, PCR reaction system: taqman Gene Expression MasterMix 12.5.5 μL; ddH 2 O8 μl; 1. Mu.L of the upstream primer (10. Mu. Mol/L); 1. Mu.L of a downstream primer (10. Mu. Mol/L); 0.5. Mu.L of probe (10. Mu. Mol/L); template DNA 2. Mu.L. The PCR mixture was prepared. The three-step method is adopted for amplification, and the amplification conditions are set as follows: 95. preheating for 10min at the temperature of 95 ℃ for 15S at 58 ℃ for 35S, and 40 cycles.
And (3) result judgment: the amplification curve was not present or the Ct value >35 was judged negative, the amplification curve was present and the Ct value >35 was judged positive. As shown in the figures 1 and 2, the experimental results show that 3 lactobacillus rhamnosus HN001 from different sources can generate amplification curves, the Ct value is less than 35, and other test bacteria do not generate amplification curves, so that the specific primers and probes can accurately identify lactobacillus rhamnosus HN001 at the strain level.
Example 2
In this example, based on the detection of DNA of Lactobacillus rhamnosus HN001, the sensitivity of the primers and probes was examined by the following experiment.
The detection sensitivity of the primer pair and the probe in the qPCR method can be determined by detecting the specific gene RepA gene and the ferritin-like domain-containing gene of lactobacillus rhamnosus HN001.
Primer pairs and probe sequences for qPCR detection were the same as in example 1.
The main detection instrument and main reagents used were the same as in example 1.
Genomic DNA of lactobacillus rhamnosus HN001 was subjected to ten-fold specific dilution into 7 gradients, each of which was repeated 3 times. The sensitivity of the method was detected by qPCR amplification using DNA solutions of various concentration gradients as templates. When the Ct value of the sample is less than or equal to 35, the detection result is positive; if the Ct value is greater than 35 or no Ct value, the detection result is judged to be negative.
The reaction system and the reaction conditions were the same as in example 1.
As shown in FIG. 3, when the target gene is RepA, the template concentration corresponding to the qPCR amplification curve is 20 and 20×10 in order -1 、20×10 -2 、20×10 -3 、20×10 -4 、20×10 -5 Mu g/mL, when the template concentration reaches 20X 10 -6 Mu g/mL, no significant amplification of template and Ct value>35, it was determined that the target gene, that is, lactobacillus rhamnosus HN001 could not be detected, and therefore, the template concentration of lactobacillus rhamnosus HN001 could be detected as 2×10 -5 Mu g/mL, namely the detection sensitivity of the qPCR method is 20 multiplied by 10 of the template concentration -5 μg/mL。
As shown in FIG. 4, when the target gene is ferritin-like domain-containing, the template concentration corresponding to the qPCR amplification curve is 20 and 20×10 in order -1 、20×10 -2 、20×10 -3 Mu g/mL, when the template concentration reaches 20X 10 -4 Mu g/mL, no significant amplification of template and Ct value>35, it was determined that the target gene, that is, lactobacillus rhamnosus HN001 could not be detected, and therefore, the template concentration of lactobacillus rhamnosus HN001 could be detected as 20×10 -3 Mu g/mL, namely the detection sensitivity of the qPCR method is 2X 10 of the template concentration -3 μg/mL。
Claims (7)
1. Two primer probes for identifying lactobacillus rhamnosus HN001, wherein a primer with a RepA gene as a target sequence is used as a forward primer 5'-GCTTAGCTTTCTCTACTGGCGA-3'; a reverse primer 5'-GTTTGCACCAGATCCCCTCA-3'; probe 5'-FAM-AAGCGCCAAGCCTTACGTGAT-BHQ1-3'; a primer taking ferritin-like domain-containing as a target sequence is taken as a forward primer 5'-CATCCAAGCCTTCTCGTGGT-3'; reverse primer 5'-ACAACATTTGGTTGGCCTGCF-3'; probe 5'-VIC-GCAAGGCCTGCAGAGTAGCGA-BHQ1-3'. The real-time fluorescence PCR method is a Taqman fluorescence probe method.
2. Two specific primers and probes for identifying lactobacillus rhamnosus HN001 are used, DNA of a sample is used as a template, qPCR amplification is carried out by using the two groups of primer probes according to the method 1, whether the sample is lactobacillus rhamnosus HN001 is judged according to whether an amplification curve appears, and the method is not used for disease diagnosis and treatment.
3. The method according to claim 2, wherein the sample is amplified by qPCR, an amplification curve is generated after the reaction is completed, and a positive result is determined if Ct value is < 35. And judging the result according to the qPCR amplification curve after the reaction is finished.
4. A kit for detecting lactobacillus rhamnosus HN001 by a qPCR method, wherein the kit comprises the primer pair and probe of claim 1.
5. A method for detecting lactobacillus rhamnosus HN001 by a qPCR method, the method comprising the use of the primer pair and probe of any of claims 1 or the kit of claim 4, which is not used for disease diagnosis and treatment purposes.
6. The method of claim 2, comprising:
(a) Providing conditions of a qPCR reaction;
(b) Using the primer pair and probe of claim 1 or the kit of claim 4, a nucleic acid amplification reaction and detection of an amplified product are performed by a qPCR method.
7. Use of the primers and probes of any one of claim 1 or the kit of claim 4 for detecting lactobacillus rhamnosus HN001, which is not used for disease diagnosis and treatment purposes.
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