CN110317891A - For detecting primer sets, reagent, kit, application and the detection method of Lactobacillus rhamnosus LV108 - Google Patents
For detecting primer sets, reagent, kit, application and the detection method of Lactobacillus rhamnosus LV108 Download PDFInfo
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Abstract
The present invention provides a kind of for detecting the primer sets of Lactobacillus rhamnosus LV108, reagent, kit, using and detection method, it is related to field of biotechnology, provided by the present invention for detecting the primer sets of Lactobacillus rhamnosus LV108, including the first primer pair, second primer pair, at least two pairs in third primer pair and the 4th primer pair, with the first primer pair, second primer pair, at least two pairs in third primer pair and the 4th primer pair can expand from Lactobacillus rhamnosus LV108 to apparent specific band as primer sets, the case where avoiding single primer pair detection inaccuracy from causing false positive, primer sets high specificity provided by the invention, high sensitivity, and amplification ability is stablized, it can be easy, quickly, accurately detection detection Lactobacillus rhamnosus LV108.
Description
Technical field
The present invention relates to field of biotechnology, more particularly, to a kind of for detecting the primer of Lactobacillus rhamnosus LV108
Group, reagent, kit, application and detection method.
Background technique
With the development and the accelerating rhythm of life of economic society, more and more people are in sub-health state.In recent years,
Largely studies have shown that lactic acid bacteria is probiotics important in human body intestinal canal, to the microecological balance for maintaining host intestine and mention
High function of immune system plays a crucial role, and can alleviate human body sub-health status or even treatment part disease.However
Lactic acid bacteria is very big with the fermenting property, enteron aisle tolerance and prebiotic function difference of strain different strains.It is long derived from Bama of Guangxi
The probiotics Lactobacillus rhamnosus LV108 prebiotic effect of Shou Cun is prominent, but since bacterial strain identifies complex difficulty, still lacks
Quickly and effectively means quickly confirm it.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first purpose of this invention be to provide it is a kind of for detecting the primer sets of Lactobacillus rhamnosus LV108 so that
Alleviate one of the technical problems existing in the prior art less.
Second object of the present invention is to provide a kind of reagent comprising above-mentioned primer sets, be lacked in the prior art with alleviating
The technical issues of few means that quickly and effectively Lactobacillus rhamnosus LV108 is confirmed.
Third object of the present invention is to provide a kind of kit comprising above-mentioned primer sets or reagent, existing to alleviate
The technical issues of lacking the means quickly and effectively confirmed to Lactobacillus rhamnosus LV108 in technology.
Fourth object of the present invention is to provide above-mentioned primer sets, reagent or kit in identification Lactobacillus rhamnosus
Whether contain the application in Lactobacillus rhamnosus LV108 in LV108, and/or detection sample to be tested.
Of the invention the 5th is designed to provide a kind of method for detecting Lactobacillus rhamnosus LV108, can be sandlwood
The identification and detection of sugared lactobacillus LV108 provides quickly and effectively means.
The present invention provides a kind of for detecting the primer sets of Lactobacillus rhamnosus LV108, and the primer sets include following 4
To at least two pairs in primer pair:
The first primer is to the single stranded DNA including entitled LV108-1-F and LV108-1-R；
The LV108-1-F include the nucleotide sequence as shown in SEQ ID NO.1, or with shown in SEQ ID NO.1
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
The LV108-1-R include the nucleotide sequence as shown in SEQ ID NO.2, or with shown in SEQ ID NO.2
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
Second primer pair includes the single stranded DNA of entitled LV108-2-F and LV108-2-R；
The LV108-2-F include the nucleotide sequence as shown in SEQ ID NO.3, or with shown in SEQ ID NO.3
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
The LV108-2-R include the nucleotide sequence as shown in SEQ ID NO.4, or with shown in SEQ ID NO.4
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
Third primer pair includes the single stranded DNA of entitled LV108-3-F and LV108-3-R；
The LV108-3-F include the nucleotide sequence as shown in SEQ ID NO.5, or with shown in SEQ ID NO.5
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
The LV108-3-R include the nucleotide sequence as shown in SEQ ID NO.6, or with shown in SEQ ID NO.6
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
4th primer pair includes the single stranded DNA of entitled LV108-4-F and LV108-4-R；
The LV108-4-F include the nucleotide sequence as shown in SEQ ID NO.7, or with shown in SEQ ID NO.7
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
The LV108-4-R include the nucleotide sequence as shown in SEQ ID NO.8, or with shown in SEQ ID NO.8
Nucleotide sequence has the nucleotide sequence of at least 80% identity.
Further, the primer sets include the first primer to, the second primer pair, third primer pair and the 4th primer pair.
Further, the first primer is 950-1050bp to the size of amplified production；And/or
The size of the second primer pair amplifies product is 1250-1350bp；And/or
The size of the third primer pair amplifies product is 1250-1350bp；And/or
The size of the 4th primer pair amplifies product is 1150-1250bp.
Further, the first primer is 1000bp to the size of amplified production；And/or
The size of the second primer pair amplifies product is 1300bp；And/or
The size of the third primer pair amplifies product is 1300bp；And/or
The size of the 4th primer pair amplifies product is 1200bp.
The present invention also provides a kind of for detecting the reagent of Lactobacillus rhamnosus LV108, and the reagent includes above-mentioned
Primer sets.
The present invention also provides a kind of for detecting the kit of Lactobacillus rhamnosus LV108, and the kit includes upper
The primer sets stated or above-mentioned reagent.
The present invention also provides above-mentioned primer sets or above-mentioned reagents at following (a) and/or (b) in application:
(a) Lactobacillus rhamnosus LV108 is identified；
(b) it detects in sample to be tested and whether contains Lactobacillus rhamnosus LV108.
Further, the sample to be tested includes more bacterial strain mixed cultures, fermented product or metabolin.
In addition, the present invention also provides a kind of methods for detecting Lactobacillus rhamnosus LV108, with the genome of strain to be tested
DNA is template, PCR amplification is carried out using above-mentioned primer sets or above-mentioned reagent or above-mentioned kit, if amplified production
In contain DNA fragment specific, then the strain to be tested includes Lee's sugar lactobacillus LV108.
Further, the first primer is 950-1050bp to the size of DNA fragment specific in amplified production；With/
Or,
The size of DNA fragment specific is 1250-1350bp in the second primer pair amplifies product；And/or
The size of DNA fragment specific is 1250-1350bp in the third primer pair amplifies product；And/or
The size of DNA fragment specific is 110-1250bp in the 4th primer pair amplifies product.
The present invention is by carrying out genome sequencing to Lactobacillus rhamnosus LV108, by the genome measured and rhamnose
Lactobacillus rhamnosus GG and Lc705 genome carry out gene family analysis, obtain the peculiar gene of Lactobacillus rhamnosus LV108, and basis
The sequence of peculiar gene and its adjacent area provide for detecting the primer sets of Lactobacillus rhamnosus LV108, including first draws
Object to, at least two pairs in the second primer pair, third primer pair and the 4th primer pair, with the first primer to, the second primer pair,
Three-primer to and the 4th primer pair at least two pairs can expand from Lactobacillus rhamnosus LV108 to apparent as primer sets
Specific band avoids single primer pair from detecting the case where inaccuracy causes false positive, primer group-specific provided by the invention
By force, high sensitivity, and amplification ability is stablized, and detection Lactobacillus rhamnosus LV108 can easy, be quickly and accurately detected.
The method of detection Lactobacillus rhamnosus LV108 provided by the invention, using the genomic DNA of strain to be tested as template,
PCR amplification, if containing DNA fragment specific in amplified production, the bacterium to be measured are carried out using primer sets provided by the invention
Strain includes Lee's sugar lactobacillus LV108.This method is easy to operate, and simple process, clinical application range is wide, also, the application present invention
The primer sets of offer are detected, and with high specificity, high sensitivity, reproducible, detection cycle is short and visual result etc. is excellent
Point can provide quickly and effectively means for the identification and detection of Lactobacillus rhamnosus LV108.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the primer sets that provide of the embodiment of the present invention 1 to Lactobacillus rhamnosus LV108 maternal plant and reference strain LGG
PCR amplification map；
Fig. 2A is the amplification figure for the primer sets of the same race lactic acid bacteria DNA non-to Lactobacillus rhamnosus that the embodiment of the present invention 2 provides
Spectrum；
Fig. 2 B be the primer sets that provide of the embodiment of the present invention 3 to Lactobacillus rhamnosus different strains (Bm01, F, grx10,
Grx19) the AFLP system of DNA, wherein hsryfm1301 is the reference strain of design of primers, belongs to Lactobacillus rhamnosus；
Fig. 2 C be the primer sets that provide of the embodiment of the present invention 3 to Lactobacillus rhamnosus different strains (SP1,1505, LY0,
BG, Bm03) DNA AFLP system；
Fig. 2 D is the primer sets that provide of the embodiment of the present invention 3 to Lactobacillus rhamnosus different strains (LV-1,1-5-1m) DNA
AFLP system；
Fig. 2 E is AFLP system of the primer sets that provide of the embodiment of the present invention 3 to commercially available bacterium powder product (Bao Dile)；
Fig. 3 is the AFLP system that the primer sets that the embodiment of the present invention 4 provides produce bacterial strain to Lactobacillus rhamnosus LV108,
Wherein, sample a, b, c, d, e are produced on December 27th, 2015, on April 20th, 2016,2016 on May 29,2016 years respectively
The product in October 28 and the January in 2018 of 5 batches on the 31st.
Specific embodiment
Unless otherwise defined herein, the scientific and technical terms used together with the present invention should have ordinary skill people
The normally understood meaning of member.The meaning and scope of term should be clear, however, in the case where any potential ambiguity, this
The definition that text provides is prior to any dictionary or external definition.In this application, unless otherwise indicated, the use of "or" means
"and/or".In addition, the use of term " includes " and other forms is non-limiting.
Generally, together with cell and tissue culture described herein, molecular biology, immunology, microbiology, science of heredity
And the nomenclature that uses of albumen and nucleic acid chemistry and hybridization and its technology be it is well known in the art that and usually using those of.
Unless otherwise indicated, methods and techniques of the invention are generally according to it is well known in the art that and as various and more specifically
Conventional method described in bibliography carries out, and the bibliography quotes and discuss from beginning to end in this specification.Enzymatic
Reaction and purification technique according to the manufacturer's instructions, such as this field usually realize or as described herein carry out.Together with this
The nomenclature and its laboratory procedure that analytical chemistry, synthetic organic chemistry and the medicine and pharmaceutical chemistry of text description use
Be with technology it is well known in the art that and usually using those of.
The present invention carries out full-length genome to the Lactobacillus rhamnosus LV108 with intestinal environment tolerance and prebiotic function
The genome measured and Lactobacillus rhamnosus GG and Lc705 genome are carried out gene family analysis by sequencing, obtain rhamnose cream
The peculiar gene of bacillus LV108；According to the sequence of peculiar gene and its adjacent area, provide a kind of for detecting rhamnose cream
The primer sets of bacillus LV108, the primer sets include at least two pairs in following 4 pairs of primer pairs:
The first primer is to the single stranded DNA including LV108-1-F and LV108-1-R；
The LV108-1-F include the nucleotide sequence as shown in SEQ ID NO.1, or with shown in SEQ ID NO.1
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
The LV108-1-R include the nucleotide sequence as shown in SEQ ID NO.2, or with shown in SEQ ID NO.2
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
Second primer pair includes the single stranded DNA of entitled LV108-2-F and LV108-2-R；
The LV108-2-F include the nucleotide sequence as shown in SEQ ID NO.3, or with shown in SEQ ID NO.3
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
The LV108-2-R include the nucleotide sequence as shown in SEQ ID NO.4, or with shown in SEQ ID NO.4
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
Third primer pair includes the single stranded DNA of entitled LV108-3-F and LV108-3-R；
The LV108-3-F include the nucleotide sequence as shown in SEQ ID NO.5, or with shown in SEQ ID NO.5
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
The LV108-3-R include the nucleotide sequence as shown in SEQ ID NO.6, or with shown in SEQ ID NO.6
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
4th primer pair includes the single stranded DNA of entitled LV108-4-F and LV108-4-R；
The LV108-4-F include the nucleotide sequence as shown in SEQ ID NO.7, or with shown in SEQ ID NO.7
Nucleotide sequence has the nucleotide sequence of at least 80% identity；
The LV108-4-R include the nucleotide sequence as shown in SEQ ID NO.8, or with shown in SEQ ID NO.8
Nucleotide sequence has the nucleotide sequence of at least 80% identity.
It, can be from Lactobacillus rhamnosus LV108 provided by the present invention for detecting the primer sets of Lactobacillus rhamnosus LV108
It expands to apparent specific band, high specificity, high sensitivity, and amplification ability is stablized, it can be easy, quickly and accurately
Detect Lactobacillus rhamnosus LV108.In addition, primer sets provided by the invention are to screen to obtain using polygenes group control experiment,
All primer sequences can accurately detect sample.
It needs to be illustrated, the present invention includes the first primer for detecting the primer sets of Lactobacillus rhamnosus LV108
To, at least two pairs in the second primer pair, third primer pair or the 4th primer pair, such as can for the first primer to and second draw
Object pair, the first primer to and third primer pair, third primer pair and the 4th primer pair, the first primer is to, the second primer pair and
Three-primer to, the second primer pair, third primer pair and the 4th primer pair or the first primer to, the second primer pair, third primer
To and the 4th primer pair.Using the first primer to, at least two pairs in the second primer pair, third primer pair and the 4th primer pair as
Primer sets can be expanded from Lactobacillus rhamnosus LV108 to apparent specific band, avoid single primer pair detection inaccuracy
The case where causing false positive improves the accuracy of testing result.
Term " identity " in the present invention refers to the similitude between sequence." identity " include with it is of the present invention
SEQ ID NO.1-SEQ ID NO.8 shown in nucleotide sequence have at least 80% (such as can be, but be not limited to
80%, 82%, 85%, 88%, 90%, 92%, the 95% or higher) nucleotide sequence of identity.
The present invention does not require the proportion between each primer pair, such as can be but be not limited to 1:1,1:1.2 or 1:
1.5, it can be adjusted according to actual use situation.
In some preferred embodiments, the primer sets include the first primer to, the second primer pair, third primer pair
With the 4th primer pair.
When primer sets provided by the invention include the first primer to, the second primer pair, third primer pair and the 4th primer pair
When, the testing result of false positive can be further avoided, the accuracy and credibility of testing result are improved.
In some preferred embodiments, the first primer is 950-1050bp to the size of amplified production, such as
It can be, but be not limited to 950bp, 980bp, 1000bp, 1020bp or 1050bp；And/or
The size of the second primer pair amplifies product is 1250-1350bp, such as can be, but be not limited to 1250bp,
1280bp, 1300bp, 1320bp or 1350bp；And/or
The size of the third primer pair amplifies product is 1250-1350bp, such as can be, but be not limited to 1250bp,
1280bp, 1300bp, 1320bp or 1350bp；And/or
The size of the 4th primer pair amplifies product is 1150-1250bp, such as can be, but be not limited to 1150bp,
1180bp, 1200bp, 1220bp or 1250bp.
It is defined by the size to amplified production, the testing result of false positive, enhancing detection can be further avoided
As a result accuracy, for example, LV108-2F/LV108-2R can be from Lactobacillus helveticus, Lactobacillus delbrueckii and Lactobacillus casei DNA
It expands to band, but size is respectively 1000bp, 4000bp and 5000bp, is compared by the size to amplified production, energy
Enough accurate judgement the above results are different from Lactobacillus rhamnosus LV108.
It should be noted that herein, "and/or" is used to indicate that one of illustrated situation or both may hair
It is raw, such as A and/or B includes (A and B) and (A or B), in the present embodiment, limits the first primer to the size of amplified production
Size for 950-1050bp perhaps the second primer pair amplifies product is 1250-1350bp or third primer pair amplifies product
Size be the size of 1250-1350bp perhaps the 4th primer pair amplifies product be 1150-1250bp or the first primer pair
The size of amplified production is 950-1050bp and the size of the second primer pair amplifies product is 1250-1350bp or third
The size of primer pair amplifies product is 1250-1350bp and the size of the 4th primer pair amplifies product is 1150-1250bp, or
Person's the first primer is 950-1050bp to the size of amplified production and the size of the second primer pair amplifies product is 1250-
The size of 1350bp and third primer pair amplifies product is the size of 1250-1350bp or the first primer to amplified production
Size for 950-1050bp and the second primer pair amplifies product is 1250-1350bp and third primer pair amplifies product
Size is 1250-1350bp and the size of the 4th primer pair amplifies product is 1150-1250bp.
In some preferred embodiments, the first primer is 1000bp to the size of amplified production；And/or
The size of the second primer pair amplifies product is 1300bp；And/or
The size of the third primer pair amplifies product is 1300bp；And/or
The size of the 4th primer pair amplifies product is 1200bp.
When the size of amplimer is exactly above-mentioned limit value, illustrate that testing result is most accurate, result credibility highest.
The present invention also provides a kind of for detecting the reagent of Lactobacillus rhamnosus LV108, and the reagent includes above-mentioned
Primer sets.
The present invention also provides a kind of for detecting the kit of Lactobacillus rhamnosus LV108, and the kit includes upper
The primer sets or reagent stated.
Based on the identical inventive concept of primer sets provided by the invention, the present invention also provides reagent or kits, therefore,
Reagent or kit provided by the invention have the beneficial effect with primer sets whole provided by the invention, and details are not described herein.
The present invention also provides above-mentioned primer sets, reagent or kits at following (a) and/or (b) in application:
(a) Lactobacillus rhamnosus LV108 is identified；
(b) it detects in sample to be tested and whether contains Lactobacillus rhamnosus LV108.
When strain to be tested is unknown strains, detected using primer sets provided by the invention, reagent or kit, and
Judge whether strain to be tested is Lee sugar lactobacillus LV108 by testing result, can reach identification Lactobacillus rhamnosus LV108's
Purpose.
When sample to be tested is the unknown samples such as more bacterial strain mixed cultures, fermented product or metabolin, the present invention is utilized
Primer sets, reagent or the kit of offer are detected, and judge whether strain to be tested is Lee's sugar lactobacillus by testing result
Whether LV108 can achieve the purpose that detect in sample to be tested containing Lactobacillus rhamnosus LV108.
It should be noted that " more bacterial strain mixed cultures " refers to the mixed culture comprising at least two bacterial strains, example
It such as can be mixed bacteria liquid.
" fermented product " refers to the product by fermentation processing, such as can be Yoghourt, cheese, fermented glutinous rice, pickles etc..
" metabolin " refers to organism metabolism object, such as can be urine or excrement etc..
In addition, the present invention also provides a kind of methods for detecting Lactobacillus rhamnosus LV108, with the genome of strain to be tested
DNA is template, PCR amplification is carried out using above-mentioned primer sets, reagent or kit, if containing specific DNA in amplified production
Segment, then the strain to be tested includes Lee's sugar lactobacillus LV108.
The method of detection Lactobacillus rhamnosus LV108 provided by the invention, easy to operate, simple process, clinical application model
It encloses extensively, also, is detected using primer sets provided by the invention, there is high specificity, high sensitivity, reproducible, detection
The advantages that period is short and visual result, can provide quickly and effectively means for the identification and detection of Lactobacillus rhamnosus LV108.
In some preferred embodiments, the first primer is to the size of DNA fragment specific in amplified production
950-1050bp；And/or
The size of DNA fragment specific is 1250-1350bp in the second primer pair amplifies product；And/or
The size of DNA fragment specific is 1250-1350bp in the third primer pair amplifies product；And/or
The size of DNA fragment specific is 110-1250bp in the 4th primer pair amplifies product.
It is defined by the size to amplified production, the testing result of false positive, enhancing detection can be further avoided
As a result accuracy.
Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only
It is used, is but should not be understood as present invention is limited in any form for being described in more detail.
The main agents information used in the embodiment of the present invention is as follows:
Primer synthesis, Sheng Gong bioengineering limited liability company (Shanghai).MRS culture medium (raw work biology, Shanghai)；Pillar
DNA extraction agent box (raw work biology, Shanghai)；RTaq archaeal dna polymerase (Takara, Dalian).Centrifuge (5424R,
Eppendorf, Germany)；PCR instrument (Mastercycler pro, eppendorf, Germany)；Electrophoresis apparatus (DYY-7C, DYCP-
31A, 61 instruments, Beijing).
The present invention is analyzed by more strain gene families, the peculiar gene of Lactobacillus rhamnosus LV108 is filtered out, peculiar
Design primer (LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3- in gene
R and LV108-4-F and LV108-4-R), and verify using PCR amplification the validity of this 4 pairs of primers.By to non-cream of the same race
Sour bacterium (lactobacillus plantarum, lactobacillus delbruockii subspecies bulgaricus, Lactobacillus helveticus, Lactobacillus casei, lactobacillus fermenti) and same
Kind different strains (hsrysm1301, grx19, LV-1, sp1,1505, LY0, BG, Bm03,1-5-1m, Bm01, F and grx10)
PCR amplification experiment, it was demonstrated that LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-
R and LV108-4-F and LV108-4-R forms the specificity expansion for being obviously different from other bacterial strains to Lactobacillus rhamnosus LV108
Increase map.
The synthesis of 1 primer of embodiment and maternal plant are examined
Genome sequencing is carried out to Lactobacillus rhamnosus LV108, is referring to bacterium with LGG, Lc705 and hsryfm1301
Strain is analyzed by more strain gene families, filters out the peculiar gene of Lactobacillus rhamnosus LV108, and set in these genes
Primer: LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R is counted, and
LV108-4-F and LV108-4-R (as shown in table 1).4 pairs of primer student on commission's work bioengineering limited liability companies (Shanghai) are closed
At.Lactobacillus rhamnosus LV108 maternal plant is crossed purifying in MRS solid medium, and picking single bacterium drops down onto 37 DEG C of MRS culture medium trainings
It supports for 24 hours, is forwarded to fresh MRS medium (2%v/v), 37 DEG C of cultures are for 24 hours；Thallus is collected, pillar DNA extraction agent box is used
(raw work biology, Shanghai) extracts the DNA of Lactobacillus rhamnosus LV108 maternal plant.It uses in rTaq archaeal dna polymerase (Takara, Dalian)
It is expanded according to DNA of 2 program of table to Lactobacillus rhamnosus LV108 maternal plant, PCR product is carried out using 1% Ago-Gel
Electrophoresis (120V, 300mA, 30min) is observed after EB dyeing.
The characteristic of 14 pairs of primers of table
The amplification condition and program of 24 pairs of primers of table
The result shows that LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-
3-R and LV108-4-F and LV108-4-R can go out clear band from the DNA cloning of Lactobacillus rhamnosus LV108 maternal plant,
Amplified band size respectively may be about 1000bp, 1300bp, 1300bp and 1200bp (Fig. 1).4 pairs of primers can not be from reference strain
The DNA cloning of LGG shows the credible result of gene family analysis to band, and 4 pairs of primers all have availability.
The non-lactic acid bacteria augmentation detection of the same race of embodiment 2
DNA is extracted and PCR method is the same as embodiment 1.In order to verify the specificity of 4 pairs of primers, first to the other bacterium of lactic acid bacteria
Kind of DNA is expanded, the results showed that LV108-1-F and LV108-1-R and LV108-3-F and LV108-3-R can not be from
Lactobacillus plantarum, lactobacillus fermenti, Lactobacillus helveticus, lactobacillus delbruockii subspecies bulgaricus and Lactobacillus casei DNA cloning
To band (Fig. 2A)；LV108-2-F and LV108-2-R can expand from Lactobacillus helveticus, Lactobacillus delbrueckii and Lactobacillus casei DNA
Increase to band, but size is respectively 1000bp, 4000bp and 5000bp, is different from Lactobacillus rhamnosus LV108；LV108-4-
F and LV108-4-R can be from lactobacillus fermenti, Lactobacillus helveticus and Lactobacillus casei DNA cloning to band, but size is respectively
2000bp, 3000bp and 4000bp are different from Lactobacillus rhamnosus LV108 (Fig. 2A).The result shows that LV108-1-F and
LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R and LV108-4-F and LV108-4-R
It is different from Lactobacillus rhamnosus LV108 to the AFLP system of several lactic acid bacterias, there is preferable differentiation effect in the level of kind.
The augmentation detection of 3 Lactobacillus rhamnosus different strains of embodiment
The purpose of the present embodiment is identify between bacterial strain, other 12 plants of mouse that 4 pairs of primer pair laboratory separation are saved
Lee's sugar lactobacillus carries out augmentation detection.Lactobacillus rhamnosus hsryfm1301 is also the reference strain of design of primers, LV108-2-F
With LV108-2-R, LV108-3-F and LV108-3-R and LV108-4-F and LV108-4-R from Lactobacillus rhamnosus
The DNA cloning of hsryfm1301 to band is respectively about 2000bp, 1500bp and 4000bp (Fig. 2 B), be different from LV108,
Further demonstrate the validity of gene family analysis.In terms of other bacterial strains, in addition to LV108-1-F and LV108-1-R is from bacterial strain
BG, V108-2-F and LV108-2-R expand from bacterial strain Bm03 and LV108-3-F and LV108-3-R from bacterial strain Bm01, F and LV-1
The band increased close to except LV108, the band that 4 pairs of primers are expanded from 12 bacterial strains be different from LV108 (Fig. 2 B, 2C,
2D), for the map of 4 bands, the AFLP system of 4 pairs of primer pairs, 12 bacterial strains is markedly different from LV108, show compared with
Good resolving effect.In order to further verify the reliability of 4 pairs of primers, the present embodiment divides from commercially available bacterium powder product (Bao Dile)
Lactobacillus rhamnosus HN001 is separated out, 4 couples of primer pair its DNA are without amplified band (Fig. 2 E).The result shows that LV108-1-F and
LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R and LV108-4-F and LV108-4-R
Lactobacillus rhamnosus LV108 is markedly different to the AFLP system of 14 plants of Lactobacillus rhamnosus, rhamnose cream bar can be used as
The mark primer of bacterium LV108.
Thus it can also be seen that LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and
LV108-3-R and LV108-4-F and LV108-4-R can also distinguish roughly laboratory separating and preserving by AFLP system
12 Lactobacillus rhamnosus strains are classified as 9 Tupu types.Bm01, F are identical with the AFLP system of LV-1,
Hsryfm1301 with 1-5-1m AFLP system is identical, remaining 7 bacterial strain all has unique AFLP system.
The augmentation detection of 4 Lactobacillus rhamnosus of embodiment production bacterial strain
In order to verify LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-
R and LV108-4-F and LV108-4-R extracts different time points to the stability of Lactobacillus rhamnosus LV108 AFLP system
Lactobacillus rhamnosus LV108 production bacterial strain DNA carry out augmentation detection.The result shows that LV108-1-F and LV108-1-R,
LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R and LV108-4-F and LV108-4-R can be from differences
Company, different batches the Lactobacillus rhamnosus LV108 bacterium powder DNA cloning of 5 batches production obtain and Lactobacillus rhamnosus
The identical map of LV108 maternal plant (Fig. 3) shows the expanding effect of 4 couples of primer pair LV108 and is not affected by long-time productive culture
It influences, it was confirmed that LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R, with
And LV108-4-F and LV108-4-R not only has specificity to the amplification of Lactobacillus rhamnosus LV108, but also has stability,
It can be used as the mark primer for confirming Lactobacillus rhamnosus LV108.
Embodiment 5
1, quickly identify Lactobacillus rhamnosus LV108 using primer sets provided by the invention
Product containing different Lactobacillus rhamnosus is cultivated, is purified, DNA is extracted, using LV108-1-F and
LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R and LV108-4-F and LV108-4-R
DNA is expanded (program is shown in Table 2), and AFLP system is compared with Lactobacillus rhamnosus LV108 maternal plant AFLP system,
Identical person is Lactobacillus rhamnosus LV108 (result is as shown in Figure 3).
2, the presence of Lactobacillus rhamnosus LV108 in primer sets provided by the invention verifying fermented product is utilized
The microorganism (concentration is not concentrated compared with Gao Shike) in concentration fermented product is collected, and it is cracked；With cracking
Liquid is template, using LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R,
And LV108-4-F and LV108-4-R carries out PCR amplification (program is shown in Table 2), if can obtain female with Lactobacillus rhamnosus LV108
The identical AFLP system of strain, shows that there are Lactobacillus rhamnosus LV108 in fermented product.
3, the presence of Lactobacillus rhamnosus LV108 in primer sets provided by the invention verifying excrement is utilized
The microorganism (concentration is not concentrated compared with Gao Shike) in concentration excrement is collected, and it is cracked；It is with lysate
Template, using LV108-1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R, and
LV108-4-F and LV108-4-R carries out PCR amplification (program is shown in Table 2), if can obtain and Lactobacillus rhamnosus LV108 maternal plant phase
Same AFLP system, shows that there are Lactobacillus rhamnosus LV108 in fecal specimens.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
SEQUENCE LISTING
<110>unified enterprise (China) Investment Co., Ltd
Kunshan Research and Development Center, Uni-President Enterprises (China) Co.,
<120>for detecting primer sets, reagent, kit, application and the detection method of Lactobacillus rhamnosus LV108
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213>artificial sequence
<400> 1
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<210> 2
<211> 25
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<213>artificial sequence
<400> 2
atcccaagtc agattgctct taaag 25
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<212> DNA
<213>artificial sequence
<400> 3
tctgtacatt gacggccgag ag 22
<210> 4
<211> 23
<212> DNA
<213>artificial sequence
<400> 4
attaggcata ctaggatcac cac 23
<210> 5
<211> 22
<212> DNA
<213>artificial sequence
<400> 5
tggtaaatgg gcactgatta ag 22
<210> 6
<211> 23
<212> DNA
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<400> 8
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Claims (10)
1. a kind of for detecting the primer sets of Lactobacillus rhamnosus LV108, which is characterized in that the primer sets include following 4 pairs
At least two pairs in primer pair:
The first primer is to the single stranded DNA including entitled LV108-1-F and LV108-1-R；
The LV108-1-F include the nucleotide sequence as shown in SEQ ID NO.1, or with nucleosides shown in SEQ ID NO.1
Acid sequence has the nucleotide sequence of at least 80% identity；
The LV108-1-R include the nucleotide sequence as shown in SEQ ID NO.2, or with nucleosides shown in SEQ ID NO.2
Acid sequence has the nucleotide sequence of at least 80% identity；
Second primer pair includes the single stranded DNA of entitled LV108-2-F and LV108-2-R；
The LV108-2-F include the nucleotide sequence as shown in SEQ ID NO.3, or with nucleosides shown in SEQ ID NO.3
Acid sequence has the nucleotide sequence of at least 80% identity；
The LV108-2-R include the nucleotide sequence as shown in SEQ ID NO.4, or with nucleosides shown in SEQ ID NO.4
Acid sequence has the nucleotide sequence of at least 80% identity；
Third primer pair includes the single stranded DNA of entitled LV108-3-F and LV108-3-R；
The LV108-3-F include the nucleotide sequence as shown in SEQ ID NO.5, or with nucleosides shown in SEQ ID NO.5
Acid sequence has the nucleotide sequence of at least 80% identity；
The LV108-3-R include the nucleotide sequence as shown in SEQ ID NO.6, or with nucleosides shown in SEQ ID NO.6
Acid sequence has the nucleotide sequence of at least 80% identity；
4th primer pair includes the single stranded DNA of entitled LV108-4-F and LV108-4-R；
The LV108-4-F include the nucleotide sequence as shown in SEQ ID NO.7, or with nucleosides shown in SEQ ID NO.7
Acid sequence has the nucleotide sequence of at least 80% identity；
The LV108-4-R include the nucleotide sequence as shown in SEQ ID NO.8, or with nucleosides shown in SEQ ID NO.8
Acid sequence has the nucleotide sequence of at least 80% identity.
2. primer sets according to claim 1, which is characterized in that the primer sets include the first primer to, the second primer
To, third primer pair and the 4th primer pair.
3. primer sets according to claim 1 or 2, which is characterized in that the first primer is to the size of amplified production
950-1050bp；And/or
The size of the second primer pair amplifies product is 1250-1350bp；And/or
The size of the third primer pair amplifies product is 1250-1350bp；And/or
The size of the 4th primer pair amplifies product is 1150-1250bp.
4. primer sets according to claim 3, which is characterized in that the first primer is to the size of amplified production
1000bp；And/or
The size of the second primer pair amplifies product is 1300bp；And/or
The size of the third primer pair amplifies product is 1300bp；And/or
The size of the 4th primer pair amplifies product is 1200bp.
5. a kind of for detecting the reagent of Lactobacillus rhamnosus LV108, which is characterized in that the reagent includes claim 1-4
Described in any item primer sets.
6. a kind of for detecting the kit of Lactobacillus rhamnosus LV108, which is characterized in that the kit includes claim
Reagent described in the described in any item primer sets of 1-4 or claim 5.
7. reagent described in primer sets according to any one of claims 1-4 or claim 5 is at following (a) and/or (b)
In application:
(a) Lactobacillus rhamnosus LV108 is identified；
(b) it detects in sample to be tested and whether contains Lactobacillus rhamnosus LV108.
8. application according to claim 7, which is characterized in that the sample to be tested includes more bacterial strain mixed cultures, hair
Ferment product or metabolin.
9. a kind of method for detecting Lactobacillus rhamnosus LV108, which is characterized in that using the genomic DNA of strain to be tested as template,
Using reagent described in the described in any item primer sets of claim 1-4 or claim 5 or reagent as claimed in claim 6
Box carries out PCR amplification, if containing DNA fragment specific in amplified production, the strain to be tested includes Lee's sugar lactobacillus
LV108。
10. according to the method described in claim 9, it is characterized in that, the first primer is to specific DNA piece in amplified production
The size of section is 950-1050bp；And/or
The size of DNA fragment specific is 1250-1350bp in the second primer pair amplifies product；
And/or
The size of DNA fragment specific is 1250-1350bp in the third primer pair amplifies product；
And/or
The size of DNA fragment specific is 110-1250bp in the 4th primer pair amplifies product.
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