CN112143820B - Molecular marker, detection primer and detection method for identifying lactobacillus plantarum and lactobacillus pentosus - Google Patents
Molecular marker, detection primer and detection method for identifying lactobacillus plantarum and lactobacillus pentosus Download PDFInfo
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Abstract
The invention discloses a molecular marker, a detection primer and a detection method for identifying lactobacillus plantarum and lactobacillus pentosus. Specific molecular markers for identifying the lactobacillus plantarum and the lactobacillus pentosus have nucleotide sequences shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or 4. The invention screens the specific molecular detection new targets of lactobacillus plantarum and lactobacillus pentosus by using a whole genome comparison analysis technology, designs a primer for the new molecular targets to carry out PCR amplification, can specifically identify two target bacteria, and does not report the molecular markers at present. Compared with a 16S rDNA PCR method and a physiological and biochemical method, the method established by the invention has higher specificity, simple operation, short detection period, no need of further sequencing and low detection cost, is particularly suitable for the rapid primary screening of L.plantarum and L.pendosus, and provides an important auxiliary identification method for food production enterprises and detection institutions.
Description
The technical field is as follows:
the invention belongs to the technical field of biology, and particularly relates to a molecular marker, a detection primer and a detection method for identifying lactobacillus plantarum and lactobacillus pentosus.
The background art comprises the following steps:
on the classification status of lactobacillus, the lactobacillus plantarum and the lactobacillus pentosus belong to the same lactobacillus plantarum group, have better inhibition effect on gram-positive and gram-negative pathogenic bacteria, and inhibit the growth of the pathogenic bacteria by nutrition competition with the pathogenic bacteria. In addition, lactobacillus plantarum can pass through the stomach and colonize the intestinal tract to play a beneficial role, and has the function of regulating the composition of intestinal microecology. In the aspect of food application, the lactobacillus plantarum is a main lactobacillus species in industrial fermented foods, is widely used for preparing fermented foods such as yoghourt, fermented sausages, bread, pickles and the like, and part of the lactobacillus plantarum can be used as a biological preservative. Lactobacillus pentosus (l.pentasus) was originally identified as lactobacillus plantarum (l.plantarum) and was then recA gene sequence difference was used to recA gene to rec-classify it as l.pentasus due to the development of molecular biology. The lactobacillus pentosus has special antibacterial and probiotic properties, plays an important role in the processes of food fermentation and preservation, and has a promoting effect on human health.
Many morphological features within lactobacillus are very similar and often cannot be accurately distinguished by traditional biochemical methods. Some strains have high homology in 16S rDNA gene sequence (L.plantarum and L.pendosus have 99% similarity), and subtype classification cannot be realized. The Uygaha et al use 16S rRNA gene, pheS and pyr G partial gene sequences to distinguish 10 strains of lactobacillus plantarum and related species thereof, use housekeeping gene pheS and pyrG partial gene sequences as molecular markers, combine corresponding sequences of related species in NCBI database to construct phylogenetic trees and compare with phylogenetic trees constructed by 16S rRNA genes, and as a result, find that L.plantarum and related species thereof cannot be distinguished based on 16S rRNA gene sequences, pheS and pyr G partial gene sequences can distinguish lactobacillus species and related species thereof, and pheS has better effect than pyrG when used for lactobacillus species endophenotyping. The gene sequences of pheS and pyrG are used in series, the classification relationship between the test strain and the reference strain is more clear, and the combined gene (pheS-pyrG) can be used as a 16S rRNA gene to assist in the rapid classification and identification of lactobacillus plantarum strains and kindred species. Some scholars design interspecific specific primers according to the common recA gene sequence of the lactobacillus plantarum, and can distinguish L.plantarum and L.pentosus in the lactobacillus plantarum by combining the nested PCR technology, but need secondary PCR amplification. In addition, the researchers used dnaK gene sequence alignment to classify lactobacillus plantarum groups.
The invention content is as follows:
the invention aims to provide a specific molecular marker for identifying lactobacillus plantarum and lactobacillus pentosus.
The invention identifies the specific molecular markers of lactobacillus plantarum and lactobacillus pentosus, and the nucleotide sequences of the specific molecular markers are shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4.
The second purpose of the invention is to provide an identification primer group for identifying lactobacillus plantarum and lactobacillus pentosus, which is more than one pair of primer combination in the following 7 pairs of primers:
_01473-Plant10-F：5'-TGCGACTGGCGATCATTTTG-3'
_01473-Plant10-R：5'-CACGATAACGGCCGCAATTT-3'；
_01473-Plant4-F：5'-TCTCGCATACGCGACTGAAA-3'
_01473-Plant4-R：5'-ACCCCGCTCCGAAATAATGG-3'；
pspA_1-plant1-F：5'-CGGCAACTACCTGTTAGCCA-3'
pspA_1-plant1-R：5'-TCGGTCGGCTTTAGTATCGC-3'；
pspA_1-plant3-F：5'-ATTAGCTTCGCGGATCGGTT-3'
pspA_1-plant3-R：5'-ACGCTCGTATTAGCTGGCTC-3'；
Pentosus_00679-2-F：5'-CCCGTCTTTAAACCGTTCGC-3'
Pentosus_00679-2-R：5'-CAACGCAGCTTGGTGTTTGA-3'；
Pentosus_00679-4-F：5'-GGTCTATTCCCCAGTGACGC-3'
Pentosus_00679-4-R：5'-CGTATTTGGCAACGCAGCTT-3'；
Pentosus_01615-8-F：5'-GCTCGTGTGGATGCTACTGA-3'
Pentosus_01615-8-R：5'-AATGGTCGACCTTCGAGTGC-3'。
the third purpose of the invention is to provide a kit for identifying lactobacillus plantarum and lactobacillus pentosus, which comprises a PCR reaction reagent and the identification primer set.
It is a fourth object of the present invention to provide a method for identifying lactobacillus plantarum and lactobacillus pentosus for non-disease diagnostic and therapeutic purposes, comprising the steps of:
extracting genome DNA of a strain to be detected, taking the identification primer group as an amplification primer, respectively carrying out PCR reaction, observing whether an amplification product exists or not, judging whether the amplification product is a target bacterium or not, if a single band with an expected size appears, the strain is a positive strain, and if the single band does not appear, the strain does not belong to the target bacterium.
Specifically, primers-01473-Plant 10-F, _01473-Plant10-R are used for PCR amplification and electrophoresis detection; carrying out PCR amplification on primers-01473-Plant 4-F, _01473-Plant4-R, primers pspA _1-Plant1-F, pspA _1-Plant1-R, primers pspA _1-Plant3-F and pspA _1-Plant3-R to respectively amplify single target bands of 280bp, 214bp, 299bp and 410bp to identify the target bands as lactobacillus plantarum; single bands with expected sizes of 417bp, 376bp and 233bp respectively appear on primers Pentosus _00679-2-F, Pentosus _00679-2-R, Pentosus _00679-4-F, Pentosus _00679-4-R, Pentosus _01615-8-F and Pentosus _01615-8-R, and the result is identified as the lactobacillus Pentosus.
The PCR reaction system comprises the following components: upstream and downstream target DNA specific primers with concentration of 10. mu. mol/L1.0. mu.L, Taq DNA polymerase 0.5. mu.L 1.5U, MgCl 25mmol/L 22 μ L of 10mmol/L dNTP 2 μ L, 10 XPCR buffer 2.5 μ L, DNA template 2.0 μ L, make up sterile deionized water 25 μ L.
The PCR reaction program is pre-denatured at 95 ℃ for 5 min; denaturation at 95 deg.C for 1min, annealing at 55-58 deg.C for 45s, extension at 72 deg.C for 1min, and amplification for 35 cycles; finally, extension is carried out for 10min at 72 ℃.
The invention screens the specific molecular detection new targets of lactobacillus plantarum and lactobacillus pentosus by using a whole genome comparison analysis technology, designs a primer for the new molecular targets to carry out PCR amplification, can specifically identify two target bacteria, and does not report the molecular markers at present. Compared with a 16S rDNA PCR method and a physiological and biochemical method, the method established by the invention has higher specificity, simple operation, short detection period, no need of further sequencing and low detection cost, is particularly suitable for the rapid primary screening of L.plantarum and L.pendosus, and provides an important auxiliary identification method for food production enterprises and detection institutions.
Description of the drawings:
FIG. 1 is an electrophoretogram of the amplification product of primer-01473-plant 4, in which 01500 bp marker, 1 negative control, 2-5plant Lactobacillus plantarum;
FIG. 2 is an electrophoretogram of the amplification product of primer-01473-plant 10, in which 01500 bp marker, 1 negative control, 2-5plant Lactobacillus plantarum;
FIG. 3 is an electrophoretogram of the amplification product of primer pspA _1-plant1, in which 01500 bp marker, 1 negative control, 2-5plant Lactobacillus plantarum;
FIG. 4 is an electrophoretogram of the amplification product of primer pspA _1-plant3, in which 01500 bp marker, 1 negative control, 2-5plant Lactobacillus plantarum;
FIG. 5 is an electrophoretogram of primers amplified by primers 01473-plant4 (upper left row), _01473-plant10 (upper right row), pspA _1-plant1 (lower left row), pspA _1-plant3 (lower right row), 1plant Lactobacillus plantarum, 2fer Lactobacillus fermentum (non-target band), 3acid Lactobacillus acidophilus, 4rh Lactobacillus rhamnosus, 5pen Lactobacillus pentosus, 6pa Lactobacillus paracasei, 7 monocytogenes Listeria CMCC54004, 8 Staphylococcus aureus ATCC29213, 9 Bacillus cereus CMCC63301, 10 Streptococcus faecalis, 11 Escherichia coli O157ATCC35150, 12 Escherichia coli ATCC25922, 01500 bp marker, respectively;
FIG. 6 is an amplification electrophoretogram of primers Pentosus _01615-8, wherein 01500 bp marker, 1-6pe Lactobacillus Pentosus, 7plant Lactobacillus plantarum, 8fer Lactobacillus fermentum, 9acid Lactobacillus acidophilus, 10rh Lactobacillus rhamnosus, 11cur Lactobacillus curvatus, 12leu Leuconostoc mesenteroides, 13helv Lactobacillus helveticus, 14pa Lactobacillus paracasei, 15 monocyte hyperplasia Listeria CMCC54004, 16 Bacillus cereus CMCC63301, 17 Streptococcus faecalis, 18 Staphylococcus aureus ATCC29213, 19 Escherichia coli O157ATCC35150, 20 negative control;
FIG. 7 is an amplification electrophoretogram of primer Pentosus _00679-2, negative controls of 01500 bp marker, 1-6pe Lactobacillus Pentosus, 7plant Lactobacillus plantarum, 8fer Lactobacillus fermentum, 9acid Lactobacillus acidophilus, 10rh Lactobacillus rhamnosus, 11cur Lactobacillus curvatus, 12leu Leuconostoc mesenteroides, 13helv Lactobacillus helveticus, 14pa Lactobacillus paracasei, 15 monocyte hyperplasia Listeria CMCC54004, 16 Bacillus cereus CMCC63301, 17 Streptococcus faecalis, 18 Staphylococcus aureus ATCC29213, 19 Escherichia coli O157ATCC35150, 20;
FIG. 8 is an amplification electrophoretogram of primers Pentosus _00679-4, negative controls of 01500 bp marker, 1-6pe Lactobacillus Pentosus, 7plant Lactobacillus plantarum, 8fer Lactobacillus fermentum, 9acid Lactobacillus acidophilus, 10rh Lactobacillus rhamnosus, 11cur Lactobacillus curvatus, 12leu Leuconostoc mesenteroides, 13helv Lactobacillus helveticus, 14pa Lactobacillus paracasei, 15 monocyte hyperplasia Listeria CMCC54004, 16 Bacillus cereus CMCC63301, 17 Streptococcus faecalis, 18 Staphylococcus aureus ATCC29213, 19 Escherichia coli O157ATCC35150, 20;
FIG. 9 shows the identification of Lactobacillus plantarum in cheese (sample + isolate), electrophoretogram of each primer amplification, M:2000bp marker, 1-2: 01473-Plant4, 3-4: pspA _1-Plant1, 5-6: 01473-Plant10, 7-8: pspA _1-Plant 3;
FIG. 10 shows the PCR amplification chart for the identification of Lactobacillus Pentosus in yogurt (sample + isolate), M:2000bp marker, 1-2: Pentosus _00679-4, 3-4: Pentosus _01615-8, and 5-6: Pentosus _ 00679-2.
The specific implementation mode is as follows:
the following is a further description of the invention and is not intended to be limiting.
(1) Sequence screening
The complete genome sequences of lactobacillus plantarum, lactobacillus pentosus and closely related species downloaded from the NCBI database. Performing Pan-genome Analysis by using an MP method in software (Pan-Genomics Analysis Pipeline, PGAP), and processing an Analysis result by using a local Perl script to obtain core gene and non-essential gene information of all strains. And (3) screening the special non-essential genes of the strains according to the pan-genomic analysis result, extracting the special non-core gene protein sequences of the lactobacillus and the related species, and respectively comparing the sequences with a protein total library of the lactobacillus and a NCBI non-redundant protein database (NR) by local Blast. Removing the sequence of the matched consensus protein, and screening out the specific marker genes of the lactobacillus plantarum and the lactobacillus pentosus. And (3) verifying that the screened molecular target belongs to a specific nucleotide sequence of the target bacteria through database blast comparison, and other non-target bacteria do not contain the gene fragment, which shows that the screened gene fragment has high specificity. The total 4 specific target fragments (2 lactobacillus plantarum and 2 lactobacillus pentosus) were screened and compared with the literature, and the same molecular markers as in the present study were not found.
Therefore, the specific molecular detection targets of lactobacillus plantarum and lactobacillus pentosus are obtained by comparing and screening the whole genome sequences of lactobacillus plantarum, lactobacillus pentosus and related species in the NCBI database, and comprise:
1. the nucleotide sequence of the target sequence of the lactobacillus plantarum is shown as SEQ ID NO.1 and SEQ ID NO. 2;
2. a target sequence of lactobacillus pentosus, the nucleotide sequence of which is shown in SEQ ID NO.3 and 4;
(2) primer design
And 7 sets of primers are provided for identifying the detection primer group of the lactobacillus plantarum and the lactobacillus pentosus by PCR, and the detection primer group has specificity to a new molecular target of a target bacterium.
TABLE 1 Lactobacillus plantarum primer sequences
TABLE 2 Lactobacillus pentosus primer sequences
(3) Identification method
A specific PCR identification method for Lactobacillus plantarum and Lactobacillus pentosus is provided, which uses a new molecular target as a target gene, selectively amplifies the thallus DNA by using the detection primer group through a PCR method, and confirms whether an amplification product exists or not.
The method comprises the following specific steps:
(1) strain DNA extraction
Recovering the strain to be identified in lactobacillus MRS broth for 24h-48h, centrifuging 1mL of bacterial culture at 12000r/min for 5min, discarding the supernatant, collecting the thallus, adding 50 μ L of lysate of the bacterial DNA extraction kit, fully suspending and uniformly mixing, centrifuging in 100 ℃ water bath for 15min, ice bath for 3min, 12000r/min for 10min, and taking the supernatant for later use, wherein the supernatant contains bacterial DNA and serves as template DNA.
(2) PCR amplification
PCR adopts 25 muL reaction system, wherein the concentration is 1.0 muL of upstream and downstream specific primers of 10 mumol/L target DNA, 0.5 muL of 1.5U Taq DNA polymerase, 25mmol/L MgCl 22 μ L of dNTP 2 μ L of 10mmol/L, 2.5 μ L of 10 XPCR buffer, 2.0 μ L of DNA template, and sterile deionized water to make up 25 μ L. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 1min, annealing at 58 ℃ for 45s, extension at 72 ℃ for 1min, and amplification for 35 cycles; finally, the extension is carried out for 10min at 72 ℃ and the product is stored at 4 ℃.
(3) Detection of PCR reaction product
The amplified PCR product was detected by electrophoresis on 1.5% agarose gel, and the result was observed by UVI of gel imaging analysis system and photographed. And observing whether the amplification product exists or not, and judging whether the amplification product is the target bacterium or not. If the amplification shows a single target band with the expected size, the strain is indicated as a positive strain, and if the target band with the expected size does not appear, the strain does not belong to the target strain.
Example 1: lactobacillus plantarum identification
1. Extraction of bacterial DNA
Centrifuging 1mL of bacterial culture at 12000r/min for 5min, discarding the supernatant, collecting thallus, adding 50 μ L of lysate of the bacterial DNA extraction kit, fully suspending and uniformly mixing, centrifuging at 100 ℃ for 15min in water bath, carrying out ice bath for 3min, and 12000r/min for 10min, and taking the supernatant for later use, wherein the supernatant contains bacterial DNA and serves as template DNA.
2. PCR amplification
PCR adopts 25 muL reaction system, wherein the concentration is 1.0 muL of upstream and downstream specific primers of 10 mumol/L target DNA, 0.5 muL of 1.5U Taq DNA polymerase, 25mmol/L MgCl 22 μ L of dNTP 2 μ L of 10mmol/L, 2.5 μ L of 10 XPCR buffer, 2.0 μ L of DNA template, and sterile deionized water to make up 25 μ L. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 1min, annealing at 58 ℃ for 45s, extension at 72 ℃ for 1min, and amplification for 35 cycles; finally, the extension is carried out for 10min at 72 ℃ and the cells are stored at 4 ℃.
3. Results of electrophoretic observations
And detecting the PCR product after the amplification is finished by using 1.5% agarose gel electrophoresis, and carrying out UVI observation and photographing by using a gel imaging analysis system. As shown in FIGS. 1, 2, 3, 4 and 5, primers-01473-Plant 10-F, _01473-Plant 10-R; the primers-01473-Plant 4-F, _01473-Plant4-R, the primers pspA _1-Plant1-F, pspA _1-Plant1-R, the primers pspA _1-Plant3-F and pspA _1-Plant3-R are subjected to PCR amplification to respectively amplify single target bands of 280bp, 214bp, 299bp and 410bp to identify the lactobacillus plantarum. The identification results are consistent with the identification results of MALDI-TOF mass spectrum, and the data of the method are proved to be reliable.
Example 2: identification of Pentobacillus
1. Extraction of bacterial DNA
Centrifuging 1mL of bacterial culture at 12000r/min for 5min, discarding the supernatant, collecting thallus, adding 50 μ L of lysate of the bacterial DNA extraction kit, fully suspending and uniformly mixing, centrifuging at 100 ℃ for 15min in water bath, carrying out ice bath for 3min, and 12000r/min for 10min, and taking the supernatant for later use, wherein the supernatant contains bacterial DNA and serves as template DNA.
2. PCR amplification
PCR adopts 25 muL reaction system, wherein the concentration is 1.0 muL of upstream and downstream specific primers of 10 mumol/L target DNA, 0.5 muL of 1.5U Taq DNA polymerase, 25mmol/L MgCl 22 μ L of dNTP 2 μ L of 10mmol/L, 2.5 μ L of 10 XPCR buffer, 2.0 μ L of DNA template, and sterile deionized water to make up 25 μ L. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 1min, annealing at 58 ℃ for 45s, extension at 72 ℃ for 1min, and amplification for 35 cycles; finally, the extension is carried out for 10min at 72 ℃ and the cells are stored at 4 ℃.
3. Results of electrophoretic observations
And detecting the PCR product after the amplification is finished by using 1.5% agarose gel electrophoresis, and carrying out UVI observation and photographing by using a gel imaging analysis system. As shown in FIGS. 6, 7 and 8, single bands of 417bp, 376bp and 233bp with expected sizes respectively appear on primers Pentosus _00679-2-F, Pentosus _00679-2-R, Pentosus _00679-4-F, Pentosus _00679-4-R, Pentosus _01615-8-F and Pentosus _01615-8-R, and the result is the lactobacillus Pentosus, which is consistent with the identification of MALDI-TOF mass spectrometry.
Example 3: identification of Lactobacillus plantarum and Lactobacillus pentosus in actual samples
1. Sample processing
A sample of cheese and yoghourt sold in the market is taken, 1-2g of the sample is added into 10mL of MRS broth, the mixture is cultured for 48h at 37 ℃, 1mL of bacterial liquid is taken, thalli is collected by centrifugation, 50 mu LDNA lysate is added, the mixture is fully suspended and uniformly mixed, the mixture is subjected to water bath at 100 ℃ for 15min, ice bath for 3min and centrifugation at 12000r/min for 10min, and the supernatant is taken as template DNA. And inoculating an MRS plate by using an inoculating loop streak, culturing at 37 ℃ for 48h, and taking a single colony to perform DNA extraction, PCR identification and mass spectrum identification.
2. PCR amplification
PCR adopts 25 mu L reaction systemWherein the concentration is 1.0 mu L of upstream and downstream specific primers of 10 mu mol/L target DNA, 0.5 mu L of 1.5U Taq DNA polymerase and 25mmol/L MgCl 22 μ L of dNTP 2 μ L of 10mmol/L, 2.5 μ L of 10 XPCR buffer, 2.0 μ L of DNA template, and sterile deionized water to make up 25 μ L. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 1min, annealing at 58 ℃ for 45s, extension at 72 ℃ for 1min, and amplification for 35 cycles; finally, extension is carried out for 10min at 72 ℃.
3. Results of electrophoretic observations
The PCR product was detected by electrophoresis on a 1.5% agarose gel and visualized by a gel imaging analysis system UVI. As shown in FIG. 9, primers-01473-Plant 10-F, _01473-Plant 10-R; the primers-01473-Plant 4-F, _01473-Plant4-R, the primers pspA _1-Plant1-F, pspA _1-Plant1-R, the primers pspA _1-Plant3-F and pspA _1-Plant3-R are subjected to PCR amplification to respectively amplify single target bands of 280bp, 214bp, 299bp and 410bp, which indicates that the lactobacillus plantarum exists in the sample.
As shown in FIG. 10, the existence of Lactobacillus Pentosus in the sample is indicated by the presence of single bands of 417bp, 376bp and 233bp with the primers Pentosus _00679-2-F, Pentosus _00679-2-R, Pentosus _00679-4-F, Pentosus _00679-4-R, Pentosus _01615-8-F and Pentosus _01615-8-R, respectively, as expected sizes, and these identification results are consistent with the identification by MALDI-TOF mass spectrometry.
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
<120> molecular marker, detection primer and detection method for identifying Lactobacillus plantarum and Lactobacillus pentosus
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 867
<212> DNA
<213> Lactobacillus plantarum (Lactobacillus plantarum)
<400> 1
atgaaagaaa aacaagtgtt tcatatcaat ggctatattg gtctagtgtt agcaatcgca 60
tttgtgttag ttggcggttg gctggtttgg gccggtgcga ctggcgatca ttttgcgagt 120
atctttttgg gcgcattgct aattattatt gcggcctttg gggctagttc gctaacaatt 180
gtgggcccca acgaggcgcg cgtgttaaca ttttttggta agtatatcgg gacaattcgt 240
gattcagggc tgtttatgac cgttccgtta accagtaagt tttccatttc actgcgggtc 300
cgcaacttta acagtgccat tttaaaagtt aatgacctcc ggggaaatcc cgtggaaatt 360
gcggccgtta tcgtgtttaa agttgttgat accagtatgg cactctttgc agtggatgat 420
tatgaacaat ttgttgagat tcaaagtgaa tcagcggtgc ggcacgtggc ctctgaatat 480
ccatatgata cgtttgatga cgacaagaag attacgctgc ggagtaatcc aaccgaagtt 540
tctgaccggt taacggaaga acttcaagaa cggctaaatg ttgcgggggt cgagatcgtt 600
gagacacgct taacgcatct cgcatacgcg actgaaatcg ccagtgcgat gttacaacgc 660
caacaatctt cggcaattct atctgctcgc aaggtcatcg tggaaggggc cgtttcaatt 720
acggaagaca cgatcgcccg tttagagaaa gatacgggga tgcagctatc agacgataag 780
aaactgcaac tgatcaataa catgatggtc accattattt cggagcgggg tactcagcca 840
attgtgaata cttcggaagt caagtag 867
<210> 2
<211> 624
<212> DNA
<213> Lactobacillus plantarum (Lactobacillus plantarum)
<400> 2
atgacaacct tttacgtggt acgacatgga caaacagtgg ccaacgccca aaatttaaaa 60
caaggcacga ttaatacagc cgtcacgcat ttgaattcgg ttggcaagca gcaagcgcaa 120
acgttgcacg ataagtttga cattagcttc gcggatcggt tgatttgtag cccgcttgac 180
cggacccagg ccacggcggc gattttgaat caaggccggc aactacctgt tagccaggac 240
gagcggttat tggaaatttc atacggccag tgggatggac aggataacga tcagttgcta 300
gcggcctatc cgcagtactt tgatccactt ttacaggacg ttttgccaag ctacgtggag 360
gttgcgacgg atggcgagac ttttgagcac gtgcaacaac gggtagcagc atttttgaag 420
acaacagcac aagcccatcc agacgagcag atcattgtcg tgacccatgg ttttaccgtc 480
aaagccatgg ccttagcgat actaaagccg accgatccga tgagcttacc ggagccagct 540
aatacgagcg tcactaaagt aatggtggat gctgtccatg gtcgccaata tttagcatat 600
tataaccgcc tacctcaatt ctga 624
<210> 3
<211> 783
<212> DNA
<213> Lactobacillus pentosus (Lactobacillus pentosus)
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atgcaaaaac accgatttta tttaaaaggc tcggccgcgg aagtcgcctg gctcaatcgt 60
caagcagacg ctggctatca actcactgcc attcatggat gcacgtatca attcgaagcg 120
acaccgactg cgaagcacgt ggtcgctgaa tacttgccga agacgacctt agacctgatg 180
acacccgtct ttaaaccgtt cgcgacccac gtctttcacg atgatttagc agtggtctat 240
tccccagtga cgccggagca gcgggtggtc aatgatgatg cccagtaccg gttagctgct 300
tatcggcacg cgcgggacgt cgccttgaat tggctgaatg gctgggtgct cgccatctgg 360
ttattgatga gtgccgcgat cgtcttgagt tctcaattaa aagcgacacc gctattgacg 420
cggattttac tgactagtct gggtctgggc gccgctttga tcgtcttggg aatcgtgatt 480
ggtgcccgcg ccgcattgcg ctgccatcgt gaagtttgcc gtttgattcg ggtgactggt 540
gacgaccagg atgcctggaa accaactttt cacgtgctat tcaaacacca agctgcgttg 600
ccaaatacgg agcagtgggc cgacctgggg cagtggcaat taacgatgca aaaccagcag 660
ggtgattatt attttgattt aagaacgacc ctgagtgaat tagagattcg ccgcacgatt 720
gccaagctag tcgctgataa ggacttcacg gtcatgtcgt ggctcggttt gtattccatt 780
tga 783
<210> 4
<211> 738
<212> DNA
<213> Lactobacillus pentosus (Lactobacillus pentosus)
<400> 4
atggcaaaga cagcaatttg cattgtcgat caacagcgat accaagtcgt tgacggcatg 60
cgattagaag aattggaatc gggactgcgg cagatgatca tggccgattt cccgcaggcg 120
cataacagta gttttatttg tagcgagcac ctcgtccatt atcggctcgc caagatggat 180
gccatgatcg aaaatgatta tcgacaaaat gataaggtca atgcgcagct ttcgaaaatt 240
ttagcgaacc atacgtatcg ggtcgtcgat gttaacagcg aactggaaca atcgctaaca 300
ttcggccaac gggtggcgga tgccgttgca cggtttggtg ggagctgggc attcatcatt 360
tcatttgtgc tggtgatgct cgtgtggatg ctactgaatg tcttaccgat ctttagccat 420
cattttgatc cgtatccctt tatcctactg aacttgttct tgagtatggt ggcggcgatt 480
caagccccgc tgatcatgat gagtcaaaat cgggcggccg aatatgatcg cttgcaagcc 540
accaatgatt ttaaagtcaa ttcgatgtct gaagaagaaa ttcgggtgct gcactcgaag 600
gtcgaccatt tgattcaaca agacgaacca aacatgcttg agattcaaaa aatgcagaca 660
cagatgttag gtgaaattca agcccaggtc aacgaactgc ggcgactgca accaaggcgg 720
cgccggaatc aaagctag 738
Claims (6)
1. A specific molecular marker combination for identifying Lactobacillus plantarum and Lactobacillus pentosus, characterized in that the nucleotide sequence is a combination of one or both of the sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and one or both of the sequences shown as SEQ ID NO.3 and SEQ ID NO. 4.
2. An identification primer set for identifying Lactobacillus plantarum and Lactobacillus pentosus, characterized in that 1 or more than 1 group is selected from the primer sets 1, 2, 3 and 4, and 1 or more than 1 group is selected from the primer sets 5, 6 and 7:
primer set 1: _01473-Plant 10-F: 5'-TGCGACTGGCGATCATTTTG-3'
_01473-Plant10-R：5'- CACGATAACGGCCGCAATTT-3'；
Primer set 2: __01473-Plant 4-F: 5'-TCTCGCATACGCGACTGAAA-3'
_01473-Plant4-R：5'- ACCCCGCTCCGAAATAATGG-3'；
Primer set 3: pspA _1-plant 1-F: 5'-CGGCAACTACCTGTTAGCCA-3'
pspA_1-plant1-R：5'- TCGGTCGGCTTTAGTATCGC-3'；
Primer set 4: pspA _1-plant 3-F: 5'-ATTAGCTTCGCGGATCGGTT-3'
pspA_1-plant3-R：5'- ACGCTCGTATTAGCTGGCTC-3'；
Primer set 5: pentosus _ 00679-2-F: 5'-CCCGTCTTTAAACCGTTCGC-3'
Pentosus_00679-2-R：5'- CAACGCAGCTTGGTGTTTGA-3'；
Primer set 6: pentosus _ 00679-4-F: 5'-GGTCTATTCCCCAGTGACGC-3'
Pentosus_00679-4-R：5'- CGTATTTGGCAACGCAGCTT-3'；
Primer set 7: pentosus _ 01615-8-F: 5'-GCTCGTGTGGATGCTACTGA-3'
Pentosus_01615-8-R：5'- AATGGTCGACCTTCGAGTGC-3'。
3. A kit for identifying Lactobacillus plantarum and Lactobacillus pentosus, comprising PCR reaction reagents and the identification primer set of claim 2.
4. A method for identifying lactobacillus plantarum and lactobacillus pentosus for non-disease diagnostic and therapeutic purposes, comprising the steps of:
extracting genome DNA of a strain to be detected, taking the identification primer group of claim 2 as an amplification primer, respectively carrying out PCR reaction, observing whether an amplification product exists or not, judging whether the amplification product is a target bacterium or not, if a single band with an expected size appears, the strain is a positive strain, and if a target band does not appear, the strain does not belong to the target bacterium;
the single bands of the expected size are specified below:
the size of an amplified band of the strain _01473-Plant10-F/R is 280 bp;
the size of an amplified band of _01473-Plant4-F/R was 214 bp;
the size of the pspA _1-plant1-F/R amplification band is 299 bp;
the size of the amplified pspA _1-plant3-F/R band is 410 bp;
the size of a Pentosus _00679-2-F/R amplification band is 417 bp;
the size of a Pentosus _00679-4-F/R amplification band is 376 bp;
the Pentosus _01615-8-F/R amplified band size was 233 bp.
5. The method of claim 4, wherein the PCR reaction system comprises: upstream and downstream target DNA specific primers with concentration of 10. mu. mol/L1.0. mu.L, Taq DNA polymerase 0.5. mu.L 1.5U, MgCl 25mmol/L22 μ L of 10mmol/L dNTP 2 μ L, 10 XPCR buffer 2.5 μ L, DNA template 2 μ L, make up sterile deionized water 25 μ L.
6. The method of claim 4, wherein the PCR reaction is performed at 95 ℃ for 5 min; denaturation at 95 deg.C for 1min, annealing at 55-60 deg.C for 45s, extension at 72 deg.C for 1min, and amplification for 35 cycles; finally, extension is carried out for 10min at 72 ℃.
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