CN110317891B - Primer group, reagent, kit, application and detection method for detecting lactobacillus rhamnosus LV108 - Google Patents
Primer group, reagent, kit, application and detection method for detecting lactobacillus rhamnosus LV108 Download PDFInfo
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Abstract
The invention provides a primer group, a reagent, a kit, an application and a detection method for detecting lactobacillus rhamnosus LV108, and relates to the technical field of biology.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group, a reagent, a kit, an application and a detection method for detecting lactobacillus rhamnosus LV 108.
Background
With the development of economic society and the acceleration of life rhythm, more and more people are in a sub-health state. In recent years, a great deal of research shows that lactic acid bacteria are important probiotics in human intestinal tracts, play a vital role in maintaining the micro-ecological balance of host intestinal tracts and improving the functions of immune systems, and can relieve the sub-health state of human bodies and even treat partial diseases. However, the fermentation performance, intestinal tolerance and probiotic function of different strains of the same lactobacillus species are greatly different. The probiotic lactobacillus rhamnosus LV108 originated from Guangxi Bama longevity village has prominent probiotic effect, but the strain identification is complex and difficult, and a quick and effective means for quickly confirming the strain is still lacked.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first object of the present invention is to provide a primer set for detecting lactobacillus rhamnosus LV108 to alleviate at least one of the technical problems of the prior art.
The second purpose of the invention is to provide a reagent containing the primer group, so as to relieve the technical problem of the lack of a quick and effective means for identifying the lactobacillus rhamnosus LV108 in the prior art.
The third purpose of the present invention is to provide a kit comprising the primer set or the reagent, so as to alleviate the technical problem of the prior art that a rapid and effective means for identifying lactobacillus rhamnosus LV108 is lacked.
The fourth purpose of the invention is to provide the application of the primer group, the reagent or the kit in identifying the lactobacillus rhamnosus LV108 and/or detecting whether the sample to be detected contains the lactobacillus rhamnosus LV 108.
The fifth purpose of the invention is to provide a method for detecting lactobacillus rhamnosus LV108, which can provide a quick and effective means for the identification and detection of lactobacillus rhamnosus LV 108.
The invention provides a primer group for detecting lactobacillus rhamnosus LV108, which comprises at least two pairs of the following 4 pairs of primer pairs:
the first primer pair includes single-stranded DNAs having the names LV108-1-F and LV 108-1-R;
the LV108-1-F comprises a nucleotide sequence shown as SEQ ID NO.1 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 1;
the LV108-1-R comprises a nucleotide sequence shown as SEQ ID NO.2 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 2;
The second primer pair comprises single-stranded DNA named LV108-2-F and LV 108-2-R;
the LV108-2-F comprises a nucleotide sequence shown as SEQ ID NO.3 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 3;
the LV108-2-R comprises a nucleotide sequence shown as SEQ ID NO.4 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 4;
the third primer pair comprises single-stranded DNA named LV108-3-F and LV 108-3-R;
the LV108-3-F comprises a nucleotide sequence shown as SEQ ID NO.5 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 5;
the LV108-3-R comprises a nucleotide sequence shown as SEQ ID NO.6 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 6;
the fourth primer pair includes single-stranded DNAs named LV108-4-F and LV 108-4-R;
the LV108-4-F comprises a nucleotide sequence shown as SEQ ID NO.7 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 7;
the LV108-4-R comprises a nucleotide sequence shown as SEQ ID NO.8 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 8.
Further, the primer set includes a first primer pair, a second primer pair, a third primer pair, and a fourth primer pair.
Further, the size of the amplification product of the first primer pair is 950-1050 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the second primer pair is 1250-1350 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the third primer pair is 1250-1350 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the fourth primer pair is 1150-1250 bp.
Further, the size of the amplification product of the first primer pair is 1000 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the second primer pair is 1300 bp; and/or the presence of a gas in the atmosphere,
the size of the amplification product of the third primer pair is 1300 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the fourth primer pair is 1200 bp.
The invention also provides a reagent for detecting the lactobacillus rhamnosus LV108, and the reagent comprises the primer group.
The invention also provides a kit for detecting lactobacillus rhamnosus LV108, and the kit comprises the primer group or the reagent.
The invention also provides the primer group or the reagent in the following (a) and/or (b):
(a) identifying lactobacillus rhamnosus LV 108;
(b) And detecting whether the sample to be detected contains lactobacillus rhamnosus LV 108.
Further, the sample to be tested comprises a multi-strain mixed culture, a fermentation product or a metabolite.
In addition, the invention also provides a method for detecting the lactobacillus rhamnosus LV108, which takes the genome DNA of a strain to be detected as a template, and uses the primer group, the reagent or the kit to carry out PCR amplification, wherein if an amplification product contains a specific DNA fragment, the strain to be detected comprises the lactobacillus rhamnosus LV 108.
Further, the size of the specific DNA fragment in the amplification product of the first primer pair is 950-1050 bp; and/or the presence of a gas in the gas,
the size of the specific DNA fragment in the amplification product of the second primer pair is 1250-; and/or the presence of a gas in the gas,
the size of the specific DNA fragment in the amplification product of the third primer pair is 1250-; and/or the presence of a gas in the gas,
the size of the specific DNA fragment in the amplification product of the fourth primer pair is 110-1250 bp.
The invention obtains the specific gene of the lactobacillus rhamnosus LV108 by carrying out whole genome sequencing on the lactobacillus rhamnosus LV108 and carrying out gene family analysis on the measured genome and the genomes of the lactobacillus rhamnosus GG and Lc705, and provides a primer group for detecting the lactobacillus rhamnosus LV108 according to the sequence of the specific gene and the adjacent region thereof, wherein the primer group comprises at least two pairs of a first primer pair, a second primer pair, a third primer pair and a fourth primer pair, at least two pairs of the first primer pair, the second primer pair, the third primer pair and the fourth primer pair are used as primer groups, so that an obvious specific band can be amplified from lactobacillus rhamnosus LV108, the condition of false positive caused by inaccurate detection of a single primer pair is avoided, the primer group provided by the invention has strong specificity and high sensitivity, and the amplification capacity is stable, and the lactobacillus rhamnosus LV108 can be simply, conveniently, quickly and accurately detected.
According to the method for detecting the lactobacillus rhamnosus LV108, genome DNA of a strain to be detected is used as a template, the primer group provided by the invention is applied to PCR amplification, and if an amplification product contains a specific DNA fragment, the strain to be detected comprises the lactobacillus rhamnosus LV 108. The method is convenient to operate, simple in process and wide in clinical application range, and the primer group provided by the invention has the advantages of strong specificity, high sensitivity, good repeatability, short detection period, visual result and the like when being used for detection, and can provide a quick and effective means for identification and detection of lactobacillus rhamnosus LV 108.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a PCR amplification map of a primer set provided in example 1 of the present invention on a Lactobacillus rhamnosus LV108 parent strain and a reference strain LGG;
FIG. 2A is an amplification map of a primer set provided in example 2 of the present invention on the DNA of a non-homologous lactic acid bacterium of Lactobacillus rhamnosus;
FIG. 2B is an amplification map of DNA of different strains of Lactobacillus rhamnosus (Bm01, F, grx10, grx19) with the primer set provided in example 3 of the present invention, wherein hsryfm1301 is a reference strain for primer design and belongs to Lactobacillus rhamnosus;
FIG. 2C is an amplification map of DNA of different strains of Lactobacillus rhamnosus (SP1, 1505, LY0, BG, Bm03) with the primer set provided in example 3 of the present invention;
FIG. 2D is an amplification map of DNA of different strains (LV-1, 1-5-1m) of Lactobacillus rhamnosus with the primer set provided in example 3 of the present invention;
FIG. 2E is an amplification spectrum of a commercial bacterial powder product (Baodile) by using the primer set provided in example 3 of the present invention;
FIG. 3 is an amplification map of a primer set provided in example 4 of the present invention on a Lactobacillus rhamnosus LV108 production strain, wherein samples a, b, c, d and e are produced in 5 batches of products at 27/12/2015, 20/4/2016, 29/2016, 10/28/2016 and 31/1/2018, respectively.
Detailed Description
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms is not limiting.
Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.
The method comprises the steps of performing whole genome sequencing on lactobacillus rhamnosus LV108 with intestinal environment tolerance and probiotic functions, and performing gene family analysis on a measured genome and genomes of lactobacillus rhamnosus GG and Lc705 to obtain a specific gene of lactobacillus rhamnosus LV 108; according to the sequences of the unique gene and the adjacent regions thereof, a primer group for detecting lactobacillus rhamnosus LV108 is provided, and the primer group comprises at least two pairs of the following 4 pairs of primer pairs:
The first primer pair comprises single-stranded DNA of LV108-1-F and LV 108-1-R;
the LV108-1-F comprises a nucleotide sequence shown as SEQ ID NO.1 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 1;
the LV108-1-R comprises a nucleotide sequence shown as SEQ ID NO.2 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 2;
the second primer pair comprises single-stranded DNA named LV108-2-F and LV 108-2-R;
the LV108-2-F comprises a nucleotide sequence shown as SEQ ID NO.3 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 3;
the LV108-2-R comprises a nucleotide sequence shown as SEQ ID NO.4 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 4;
the third primer pair comprises single-stranded DNA named LV108-3-F and LV 108-3-R;
the LV108-3-F comprises a nucleotide sequence shown as SEQ ID NO.5 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 5;
the LV108-3-R comprises a nucleotide sequence shown as SEQ ID NO.6 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 6;
The fourth primer pair includes single-stranded DNAs having the names LV108-4-F and LV 108-4-R;
the LV108-4-F comprises a nucleotide sequence shown as SEQ ID NO.7 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 7;
the LV108-4-R comprises a nucleotide sequence shown as SEQ ID NO.8 or a nucleotide sequence with at least 80% of identity with the nucleotide sequence shown as SEQ ID NO. 8.
The primer group for detecting the lactobacillus rhamnosus LV108 provided by the invention can be used for amplifying an obvious specific band from the lactobacillus rhamnosus LV108, has strong specificity, high sensitivity and stable amplification capability, and can be used for simply, conveniently, quickly and accurately detecting the lactobacillus rhamnosus LV 108. In addition, the primer group provided by the invention is obtained by screening a multi-gene group comparison experiment, and all primer sequences can accurately detect a sample.
It should be noted that the primer set for detecting lactobacillus rhamnosus LV108 according to the present invention includes at least two pairs of the first primer pair, the second primer pair, the third primer pair or the fourth primer pair, for example, the first primer pair and the second primer pair, the first primer pair and the third primer pair, the third primer pair and the fourth primer pair, the first primer pair, the second primer pair and the third primer pair, the second primer pair, the third primer pair and the fourth primer pair, or the first primer pair, the second primer pair, the third primer pair and the fourth primer pair. At least two pairs of the first primer pair, the second primer pair, the third primer pair and the fourth primer pair are used as primer groups, so that an obvious specific band can be amplified from lactobacillus rhamnosus LV108, the condition of false positive caused by inaccurate detection of a single primer pair is avoided, and the accuracy of a detection result is improved.
The term "identity" in the present invention refers to the similarity between sequences. "identity" includes nucleotide sequences having at least 80% (e.g., may be, but is not limited to, 80%, 82%, 85%, 88%, 90%, 92%, 95% or more) identity to the nucleotide sequences represented by SEQ ID No.1-SEQ ID No.8 as described herein.
The ratio of the primer pairs is not required, and can be, but is not limited to, 1:1, 1:1.2 or 1:1.5, and can be adjusted according to actual use conditions.
In some preferred embodiments, the primer set comprises a first primer pair, a second primer pair, a third primer pair, and a fourth primer pair.
When the primer group provided by the invention comprises the first primer pair, the second primer pair, the third primer pair and the fourth primer pair, the detection result of false positive can be further avoided, and the accuracy and the credibility of the detection result are improved.
In some preferred embodiments, the size of the amplification product of the first primer pair is 950-1050bp, such as, but not limited to, 950bp, 980bp, 1000bp, 1020bp or 1050 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the second primer pair is 1250-1350bp, and for example, but not limited to 1250bp, 1280bp, 1300bp, 1320bp or 1350 bp; and/or the presence of a gas in the gas,
The size of the amplification product of the third primer pair is 1250-1350bp, for example, but not limited to 1250bp, 1280bp, 1300bp, 1320bp or 1350 bp; and/or the presence of a gas in the atmosphere,
the size of the amplification product of the fourth primer pair is 1150-1250bp, which can be, but is not limited to 1150bp, 1180bp, 1200bp, 1220bp or 1250 bp.
For example, LV108-2F/LV108-2R can be amplified to bands from DNA of lactobacillus helveticus, lactobacillus delbrueckii and lactobacillus casei, but the sizes are 1000bp, 4000bp and 5000bp respectively, and the results can be accurately judged to be different from the results of lactobacillus rhamnosus LV108 by comparing the sizes of the amplified products.
Note that "and/or" is used herein to indicate that one or both of the illustrated cases may occur, for example, where A and/or B includes (A and B) and (A or B), in this embodiment, the size of the amplification product of the first primer pair is defined as 950- The size of the amplification product of the third primer pair is 1250-1350bp, or the size of the amplification product of the first primer pair is 950-1050bp and the size of the amplification product of the second primer pair is 1250-1350bp and the size of the amplification product of the third primer pair is 1250-1350bp and the size of the amplification product of the fourth primer pair is 1150-1250 bp.
In some preferred embodiments, the first primer pair amplification product has a size of 1000 bp; and/or the presence of a gas in the atmosphere,
the size of the amplification product of the second primer pair is 1300 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the third primer pair is 1300 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the fourth primer pair is 1200 bp.
When the size of the amplification primer is just the limit value, the detection result is most accurate, and the result reliability is highest.
The invention also provides a reagent for detecting the lactobacillus rhamnosus LV108, and the reagent comprises the primer group.
The invention also provides a kit for detecting the lactobacillus rhamnosus LV108, and the kit comprises the primer group or the reagent.
Based on the same inventive concept of the primer set provided by the invention, the invention also provides a reagent or a kit, so that the reagent or the kit provided by the invention has the overall beneficial effects with the primer set provided by the invention, and the details are not repeated herein.
The invention also provides application of the primer group, the reagent or the kit in the following (a) and/or (b):
(a) identifying lactobacillus rhamnosus LV 108;
(b) and detecting whether the sample to be detected contains lactobacillus rhamnosus LV 108.
When the strain to be detected is an unknown strain, the primer group, the reagent or the kit provided by the invention is used for detection, and whether the strain to be detected is the lactobacillus rhamnosus LV108 or not is judged according to the detection result, so that the aim of identifying the lactobacillus rhamnosus LV108 can be fulfilled.
When the sample to be detected is an unknown sample such as a multi-strain mixed culture, a fermentation product or a metabolite, the primer group, the reagent or the kit provided by the invention is used for detection, and whether the strain to be detected is lactobacillus rhamnosus LV108 or not is judged according to the detection result, so that the purpose of detecting whether the sample to be detected contains lactobacillus rhamnosus LV108 or not can be achieved.
The "multi-strain mixed culture" refers to a mixed culture containing at least two strains, and may be, for example, a mixed bacterial solution.
"fermented product" refers to a product that has undergone a fermentation process, and may be, for example, yogurt, cheese, fermented glutinous rice, kimchi, etc.
"metabolite" refers to a metabolite of the body, which may be, for example, urine or feces, etc.
In addition, the invention also provides a method for detecting the lactobacillus rhamnosus LV108, which takes the genome DNA of a strain to be detected as a template, and applies the primer group, the reagent or the kit to perform PCR amplification, wherein if an amplification product contains a specific DNA fragment, the strain to be detected comprises the lactobacillus rhamnosus LV 108.
The method for detecting the lactobacillus rhamnosus LV108 provided by the invention is convenient to operate, simple in process and wide in clinical application range, and the primer group provided by the invention has the advantages of strong specificity, high sensitivity, good repeatability, short detection period, visual result and the like when being used for detection, and can provide a quick and effective means for identification and detection of the lactobacillus rhamnosus LV 108.
In some preferred embodiments, the size of the specific DNA fragment in the amplification product of the first primer pair is 950-1050 bp; and/or the presence of a gas in the atmosphere,
the size of the specific DNA fragment in the amplification product of the second primer pair is 1250-1350 bp; and/or the presence of a gas in the atmosphere,
the size of the specific DNA fragment in the amplification product of the third primer pair is 1250-1350 bp; and/or the presence of a gas in the atmosphere,
the size of the specific DNA fragment in the amplification product of the fourth primer pair is 110-1250 bp.
By limiting the size of the amplified product, false positive detection results can be further avoided, and the accuracy of the detection results is enhanced.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The main reagent information used in the examples of the present invention is as follows:
primer synthesis, bio-engineering, Inc. (Shanghai). MRS medium (biologies, shanghai); column type DNA extraction kit (Biopsis, Shanghai); rTaq DNA polymerase (Takara, Large ligation). Centrifuge (5424R, eppendorf, germany); PCR instruments (Mastercycler pro, eppendorf, Germany); electrophoresis apparatus (DYY-7C, DYCP-31A, Liuyi apparatus, Beijing).
According to the invention, through multi-strain gene family analysis, the specific gene of the Lactobacillus rhamnosus LV108 is screened out, primers (LV108-1-F, LV108-1-R, LV108-2-F, LV108-2-R, LV108-3-F, LV108-3-R, LV108-4-F and LV108-4-R) are designed in the specific gene, and the effectiveness of the 4 pairs of primers is verified by utilizing PCR amplification. LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R, as well as LV108-4-F and LV108-4-R, were confirmed to form a specific pattern amplification for Lactobacillus rhamnosus LV108 that is clearly distinguished from other strains by PCR amplification experiments on non-homologous lactic acid bacteria (Lactobacillus plantarum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus fermentum) and homologous different strains (hsrysm1301, grx19, LV-1, sp1, 1505, LY0, BG, Bm03, 1-5-1m, Bm01, F and grx 10).
Example 1 primer Synthesis and stock test
Carrying out whole genome sequencing on the lactobacillus rhamnosus LV108, taking LGG, Lc705 and hsryfm1301 as reference strains, screening out specific genes of the lactobacillus rhamnosus LV108 through multi-strain gene family analysis, and designing primers in the genes: LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R, as well as LV108-4-F and LV108-4-R (as shown in Table 1). The 4 pairs of primers were synthesized by Competition Bioengineering, Inc. (Shanghai). The Lactobacillus rhamnosus LV108 stock strain is streaked and purified in an MRS solid culture medium, a single colony is picked and cultured in the MRS culture medium at 37 ℃ for 24h, and is transferred to a fresh MRS culture medium (2% v/v) and cultured at 37 ℃ for 24 h; collecting thallus, and extracting DNA of Lactobacillus rhamnosus LV108 stock strain with column type DNA extraction kit (living organism, Shanghai). The DNA of the Lactobacillus rhamnosus LV108 stock strain was amplified using rTaq DNA polymerase (Takara, Dalian) according to the procedure of Table 2, and the PCR products were electrophoresed using 1% agarose gel (120V,300mA,30min), followed by observation after EB staining.
TABLE 14 Properties of the pairs of primers
TABLE 24 amplification conditions and procedures for the primers
The results show that LV108-1-F, LV108-1-R, LV108-2-F, LV108-2-R, LV108-3-F, LV108-3-R and LV108-4-F and LV108-4-R can amplify clear bands from the DNA of the Lactobacillus rhamnosus LV108 mother strain, and the amplified bands are about 1000bp, 1300bp and 1200bp respectively (FIG. 1). None of the 4 primers amplified a band from the DNA of the reference strain LGG, indicating that the results of the gene family analysis are reliable and that the 4 primers are available.
Example 2 amplification assay for non-homogeneous lactic acid bacteria
The DNA extraction and PCR were performed as in example 1. In order to verify the specificity of the 4 pairs of primers, the DNA of other strains of lactic acid bacteria was amplified first, and the results showed that neither LV108-1-F nor LV108-1-R nor LV108-3-F nor LV108-3-R could be amplified to bands from the DNA of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus delbrueckii subsp.bulgaricus and Lactobacillus casei (FIG. 2A); LV108-2-F and LV108-2-R can be amplified to bands from Lactobacillus helveticus, Lactobacillus delbrueckii and Lactobacillus casei DNA, but the sizes are respectively 1000bp, 4000bp and 5000bp, which are all different from Lactobacillus rhamnosus LV 108; LV108-4-F and LV108-4-R were able to amplify to bands from L.fermentum, L.helveticus and L.casei DNA, but were 2000bp, 3000bp and 4000bp in size, respectively, all different from L.rhamnosus LV108 (FIG. 2A). The result shows that the amplification patterns of LV108-1-F, LV108-1-R, LV108-2-F, LV108-2-R, LV108-3-F, LV108-3-R and LV108-4-F and LV108-4-R on several lactic acid bacteria are different from that of Lactobacillus rhamnosus LV108, and the strain level has better distinguishing effect.
Example 3 amplification assay of different strains of Lactobacillus rhamnosus
The purpose of this example is to perform the identification between strains, and 4 pairs of primers were used to perform amplification detection on other 12 strains of lactobacillus rhamnosus isolated and stored in the laboratory. Lactobacillus rhamnosus hsryfm1301 is also a reference strain for primer design, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R, as well as LV108-4-F and LV108-4-R bands of about 2000bp, 1500bp and 4000bp, respectively, obtained from the DNA amplification of Lactobacillus rhamnosus hsryfm1301 (FIG. 2B), are different from LV108, further confirming the effectiveness of gene family analysis. On the other strain side, the bands amplified from 12 strains by 4 pairs of primers are different from LV108 (FIG. 2B,2C,2D) except that the bands amplified from strains BG, V108-2-F and LV108-2-R by LV108-1-F and LV108-2-R by Bm03 and strains Bm01, F and LV108-3-R by LV108-3-F and LV108-1-R are close to LV108, and the amplification patterns of 12 strains by 4 pairs of primers are remarkably different from LV108 in the case of the 4-band patterns, thus showing better resolution effect. To further verify the reliability of the 4 primers, lactobacillus rhamnosus HN001 was isolated from commercial bacterial powder product (portulare) in this example, and no amplified band was observed in any of the 4 primers (fig. 2E). The result shows that the amplification maps of LV108-1-F, LV108-1-R, LV108-2-F, LV108-2-R, LV108-3-F, LV108-3-R and LV108-4-F and LV108-4-R on 14 strains of lactobacillus rhamnosus are all obviously different from that of lactobacillus rhamnosus LV108, and the primers can be used as marker primers of the lactobacillus rhamnosus LV 108.
It can also be seen from this that LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R, as well as LV108-4-F and LV108-4-R, also enable a rough differentiation of the 12 Lactobacillus rhamnosus strains deposited in the laboratory by amplifying the profiles, dividing them into 9 profile types. The amplification patterns of Bm01, F and LV-1 are the same, the amplification patterns of hsryfm1301 and 1-5-1m are the same, and the other 7 strains have unique amplification patterns.
Example 4 amplification assay of Lactobacillus rhamnosus producing strains
In order to verify the stability of LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R, as well as LV108-4-F and LV108-4-R on the amplification map of Lactobacillus rhamnosus LV108, the DNA of the Lactobacillus rhamnosus LV108 producing strain at different time points was extracted for amplification testing. The results show that LV108-1-F, LV108-1-R, LV108-2-F, LV108-2-R, LV108-3-F, LV108-3-R and LV108-4-F and LV108-4-R can be amplified from lactobacillus rhamnosus LV108 powder DNA produced by different companies and different batches of 5 batches to obtain the same map as the lactobacillus rhamnosus LV108 parent strain (figure 3), show that the amplification effect of 4 pairs of primers on LV108 is not influenced by long-time production culture, and prove that LV108-1-F, LV108-1-R, LV108-2-F, LV108-2-R, LV108-3-F, LV108-3-R and LV108-4-F and LV108-4-R not only have specificity on the amplification of lactobacillus rhamnosus LV108, and has stability, and can be used as a marker primer for verifying lactobacillus rhamnosus LV 108.
Example 5
1. The primer group provided by the invention is used for rapidly identifying lactobacillus rhamnosus LV108
Products containing different lactobacillus rhamnosus are cultured, purified, DNA is extracted, the DNA is amplified by using LV108-1-F, LV108-1-R, LV108-2-F, LV108-2-R, LV108-3-F, LV108-3-R and LV108-4-F and LV108-4-R (the program is shown in a table 2), and the amplification map is compared with the amplification map of a lactobacillus rhamnosus LV108 mother strain, namely lactobacillus rhamnosus LV108 (the result is shown in a table 3).
2. The primer group provided by the invention is used for verifying the existence of lactobacillus rhamnosus LV108 in a fermented product
Collecting microorganisms (which may not be concentrated when the concentration is higher) in the concentrated fermentation product, and performing lysis on the microorganisms; and (3) performing PCR amplification by using the lysate as a template and using LV108-1-F, LV108-1-R, LV108-2-F, LV108-2-R, LV108-3-F, LV108-3-R and LV108-4-F and LV108-4-R (the program is shown in Table 2), wherein if the same amplification map as that of the Lactobacillus rhamnosus LV108 mother strain can be obtained, the presence of the Lactobacillus rhamnosus LV108 in a fermentation product is shown.
3. The primer group provided by the invention is used for verifying the existence of lactobacillus rhamnosus LV108 in excrement
Collecting microorganisms (which may not be concentrated when the concentration is higher) in the concentrated excrement, and cracking the microorganisms; and (3) performing PCR amplification by using the lysate as a template and using LV108-1-F, LV108-1-R, LV108-2-F, LV108-2-R, LV108-3-F, LV108-3-R and LV108-4-F and LV108-4-R (the program is shown in Table 2), wherein if the same amplification map as that of the Lactobacillus rhamnosus LV108 mother strain can be obtained, the existence of the Lactobacillus rhamnosus LV108 in the feces sample is shown.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> investment Limited of unified Enterprise (China)
KUNSHAN RESEARCH & DEVELOPMENT CENTER OF UNI-PRESIDENT (CHINA) INVESTMENT Co.,Ltd.
<120> primer set, reagent, kit, application and detection method for detecting lactobacillus rhamnosus LV108
<160> 8
<170> PatentIn version 3.5
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Claims (10)
1. A primer group for detecting Lactobacillus rhamnosus LV108, wherein the primer group comprises at least two pairs of the following 4 pairs of primer pairs:
the first primer pair comprises single-stranded DNA named LV108-1-F and LV 108-1-R;
the LV108-1-F is a nucleotide sequence shown as SEQ ID NO. 1;
The LV108-1-R is a nucleotide sequence shown as SEQ ID NO. 2;
the second primer pair comprises single-stranded DNA named LV108-2-F and LV 108-2-R;
the LV108-2-F is a nucleotide sequence shown as SEQ ID NO. 3;
the LV108-2-R is a nucleotide sequence shown as SEQ ID NO. 4;
the third primer pair comprises single-stranded DNA named LV108-3-F and LV 108-3-R;
the LV108-3-F is a nucleotide sequence shown as SEQ ID NO. 5;
the LV108-3-R is a nucleotide sequence shown as SEQ ID NO. 6;
the fourth primer pair includes single-stranded DNAs named LV108-4-F and LV 108-4-R;
the LV108-4-F is a nucleotide sequence shown as SEQ ID NO. 7;
the LV108-4-R is a nucleotide sequence shown as SEQ ID NO. 8.
2. The primer set of claim 1, wherein the primer set comprises a first primer pair, a second primer pair, a third primer pair, and a fourth primer pair.
3. The primer set as claimed in claim 1 or 2, wherein the size of the amplification product of the first primer pair is 950-1050 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the second primer pair is 1250-1350 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the third primer pair is 1250-1350 bp; and/or the presence of a gas in the gas,
The size of the amplification product of the fourth primer pair is 1150-1250 bp.
4. The primer set of claim 3, wherein the size of the amplification product of the first primer pair is 1000 bp; and/or the presence of a gas in the atmosphere,
the size of the amplification product of the second primer pair is 1300 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the third primer pair is 1300 bp; and/or the presence of a gas in the gas,
the size of the amplification product of the fourth primer pair is 1200 bp.
5. A reagent for detecting Lactobacillus rhamnosus LV108, wherein said reagent comprises the primer set of any of claims 1-4.
6. A kit for detecting Lactobacillus rhamnosus LV108, characterized in that, the kit includes the primer set of any one of claims 1-4, or the reagent of claim 5.
7. Use of the primer set according to any one of claims 1 to 4, or the reagent according to claim 5 in (a) and/or (b) below:
(a) identifying lactobacillus rhamnosus LV 108;
(b) and detecting whether the sample to be detected contains lactobacillus rhamnosus LV 108.
8. The use of claim 7, wherein the test sample comprises a multi-strain mixed culture, a fermentation product, or a metabolite.
9. A method for detecting Lactobacillus rhamnosus LV108 is characterized in that genomic DNA of a strain to be detected is used as a template, a primer set according to any one of claims 1 to 4, a reagent according to claim 5 or a kit according to claim 6 is used for PCR amplification, and if specific DNA fragments are contained in an amplification product, the strain to be detected comprises Lactobacillus rhamnosus LV 108.
10. The method as claimed in claim 9, wherein the size of the specific DNA fragment in the amplification product of the first primer pair is 950-1050 bp; and/or the presence of a gas in the atmosphere,
the size of the specific DNA fragment in the amplification product of the second primer pair is 1250-; and/or the presence of a gas in the gas,
the size of the specific DNA fragment in the amplification product of the third primer pair is 1250-; and/or the presence of a gas in the gas,
the size of the specific DNA fragment in the amplification product of the fourth primer pair is 110-1250 bp.
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