CN109652573A - For Salmonella typhimurtum or the site VNTR, detection primer group and the determination method of its single-phase bacterium mutation parting detection - Google Patents
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Abstract
The invention discloses a kind of site VNTR, detection primer group and determination methods detected for Salmonella typhimurtum or its single-phase bacterium mutation parting.The present invention is directed to Salmonella typhimurtum and its single-phase bacterium mutation detection of Salmonella Isosorbide-5-Nitrae, [5], and 12:i:- newly screens 2 sites VNTR, and is combined known 5 sites VNTR, establishes the site MLVA-2 method and the site MLVA-7 method.By a large amount of Salmonella typhimurtums and its single-phase bacterium mutation detection of Salmonella 1,4, [5], the detection verifying of 12:i:- bacterial strain, determination method of the invention can be directly to Salmonella typhimurtum and its single-phase bacterium mutation detection of Salmonella 1,4, [5], 12:i:- carries out stable parting detection, only analyze 2 genes, parting efficiency is suitable with traditional MLST classifying method, only analyzes 7 genes, and parting efficiency is higher than combination original site MLST and MLVA-5 classifying method.
Description
Technical field:
The invention belongs to biomedicine fields, and in particular to one kind is used for Salmonella typhimurtum or its single-phase bacterium mutation parting
The site VNTR, detection primer group and the determination method of detection.
Background technique:
Detection of Salmonella (Salmonella spp) is the zoonosis pathogen being of great significance on public hygienics, can
Cause many serious diseases such as typhoid fever, paratyphoid, gastroenteritis and the septicemia of humans and animals, makes the whole world by huge economic damage
It loses.Currently, the whole world has found about 2610 kinds of detection of Salmonella serotypes.Wherein, Salmonella typhimurtum (Salmonella
Enterica subsp.enterica serovar Typhimurium, STM) it is the advantage blood being present in livestock and poultry and food
Clear type, and cause the predominant serotypes of human infection.Since 21 century, a kind of emerging highly pathogenic serotype --- it is husky
Door bacterium1, 4, [5], 12:i:- is constantly detected in multiple countries, and causes food origin disease outbreak of epidemic.Detection of Salmonella1,4,
[5], 12:i:- and Salmonella typhimurtum (antigen formula:1, 4, [5], 12:i:1,2) and there is comm on antigen form, the difference of the two
It is detection of Salmonella1, 4, [5], 12:i:- has lacked the IIth phase flagellar antigen.It is multinomial that researches show that detection of Salmonella1,4,[5],12:i:-
There is very high genetic similarty with Salmonella typhimurtum, be the single-phase bacterium mutation of Salmonella typhimurtum.
Area is subject to using detection of Salmonella of the effective classifying method to the even different pathogenicities of separate sources, different serotypes
Point, to detection of Salmonella harm is traced to the source, Risk-warning and epidemic monitoring are of great significance.Molecule parting uses different molecule skills
The method of art reflection pathogenic bacteria genomic dna sequence difference has stable, accurate, quick, resolving power height and strong operability etc. excellent
Point.It is some commonly based on the molecular typing methods of nucleic acid sequence, such as Multilocus sequence typing (Multilocus sequence
Typing, MLST) and multidigit point variable number tandem repeat analysis (Multiple-locus variable-number
Tandem-repeat analysis, MLVA), it is accurate, reproducible, data exchange and sharing is realized, so that different experiments
Result between room is comparable, and is occurred in the retrospect of pathogen, the confirmation of propagation chain, the discovery of new epidemic link, system
The early-warning and predicting etc. of analysis even epidemic situation has played important function.
MLST parting be by 7 house-keeping genes relatively conservative on analysis detection of Salmonella genome (arcC, dnaN, hemD,
HisD, purE, sucA and thrA), the allele of house-keeping gene, root are obtained by DNA sequencing technology and BLAST comparison technology
The change of pathogenic bacteria gene profile is disclosed according to base mutation.The allelotype of 7 house-keeping genes forms an allele spectrum, often
A allele composes corresponding only one sequence type, i.e. ST type (Sequence type, ST).Studies have shown that MLST classifying method
By Salmonella typhimurtum and its single-phase bacterium mutation detection of Salmonella1, 4, [5], 12:i:- bacterial strain is divided into two big clusters, referred to as eBGs
(eBurstGroups), the eBG1 cluster that a big cluster is made of the main type bacterial strain such as ST19, ST34, another cluster is by ST36 type
The eBG138 cluster of bacterial strain composition (see Fig. 1).And the single-phase bacterium mutation detection of Salmonella of mouse typhus1, 4, [5], 12:i:- serotype is at present only
It is found to have the bacterial strain for being present in tri- kinds of types of ST36 of ST19, ST34 and eBG138 cluster of eBG1 cluster (see Fig. 1).
MLVA classifying method is to utilize different loci tandem repetitive sequence (Variable-number in microbial genome
Tandem-repeat, VNTR) number difference, parting is carried out to it, is widely used in the research of microorganism, especially bacterium.
MLVA method is easy to operate, quick, high resolution.Lindstedt in 2004 etc. utilizes 5 VNTR of Salmonella typhimurtum
Point-STTR9, STTR5, STTR6, STTR10, STTR3 have been successfully established the site the MLVA-5 parting side for Salmonella typhimurtum
Method, this method substantially increase the resolution capability to Salmonella typhimurtum polymorphism.
Based on the MLST method of 7 house-keeping genes, ST type not only may determine that the affiliation between sramana's bacteria strain,
Also there is corresponding relationship with detection of Salmonella serotype, compared to MLST method, MLVA provides a kind of higher parting effect of resolution ratio,
Therefore, two kinds of classifying methods of use in conjunction MLST and MLVA carry out parting to detection of Salmonella, trace to the source detection of Salmonella harm, Risk-warning
It is of great significance with epidemic monitoring.In actual operation, mouse is hurt if two kinds of classifying methods of MLST and MLVA are respectively adopted
Cold detection of Salmonella parting, will analyze the nucleic acid sequence of 12 genes, heavy workload, time-consuming, costly altogether.
Summary of the invention:
The purpose of the invention is to overcome defect in the prior art, a kind of more effective, easier, high-resolution is provided
For Salmonella typhimurtum or the site VNTR, detection primer group and the determination method of its single-phase bacterium mutation parting detection.
In view of the above-mentioned problems, the present invention screens two new sites VNTR in actual operation --- STMV1 and
STMV2 designs specific nucle primer sequence for the two sites, establish the site MLVA-2 method to Salmonella typhimurtum and
Its single-phase bacterium mutation detection of Salmonella1, 4, [5], 12:i:- carries out parting, the bacterial strain ST type in genotyping result and MLST classifying method
With certain correlation.
When the site MLVA-2 method is applied to Salmonella typhimurtum, the repetition in the site STMV1 of eBG138 cluster ST36 type bacterial strain
Number all 8, meanwhile, the types bacterial strain such as ST19, ST34, ST313, ST213, ST128, ST568 and ST302 of eBG1 cluster
The repeat number in the site STMV1 all 9.The site joint STMV2, the repeat number in the site STMV1 of the ST34 type bacterial strain of eBG1 cluster
ST19, ST313, ST213, ST128, ST568 and ST302 type bacterium of all 4, the eBG1 clusters of the repeat number of all 9 and STMV2
The repeat number all 9 in the site STMV1 of strain and the repeat number all 3 in the site STMV2.For being divided into eBG1 and eBG138 two
Big cluster and the Salmonella typhimurtum with numerous MLST types, STMV1 and STMV2 can identify two cluster bacterium of eBG1 and eBG138
ST34 type bacterial strain in strain and eBG1 cluster.
The site MLVA-2 method is applied to the single-phase bacterium mutation of mouse typhus only with tri- kinds of type bacterial strains of ST19, ST34, ST36
Detection of Salmonella1, 4, [5], 12:i:-, the repeat number all 8 in the site STMV1 of the ST36 type bacterial strain of eBG138 cluster, meanwhile, eBG1
The repeat number all 9 in the site STMV1 of ST19, ST34 type bacterial strain of cluster.The site joint STMV2, the ST34 type bacterium of eBG1 cluster
The ST19 type bacterial strain of all 4, the eBG1 clusters of repeat number of the repeat number all 9 and the site STMV2 in the site STMV1 of strain
The repeat number in the site STMV1 all 9 and the repeat number in the site STMV2 all 3.
The further primer of 7 sites VNTR STMV1, STMV2, STTR9, STTR5, STTR6, STTR10, STTR3 of combination
Combination, establishes a kind of Salmonella typhimurtum and its single-phase bacterium mutation detection of Salmonella1, 4, [5], 12:i:- parting new method ---
The site MLVA-7 method improves parting efficiency.According to each site repetitive sequence in each site pcr amplified fragment sequencing result
Number carries out parting, is as a result expressed as n-n-n-n-n-n-n.
Therefore, the first purpose of the invention is to provide one kind to be used for Salmonella typhimurtum or its single-phase bacterium mutation detection of Salmonella1, 4, [5], the site VNTR of 12:i:- parting detection, the site the VNTR number is STMV1 and STMV2；
The nucleotide sequence of the STMV1 is as shown in SEQ ID NO.1；
The nucleotide sequence of the STMV2 is as shown in SEQ ID NO.2.
A second object of the present invention is to provide one kind to be used for Salmonella typhimurtum or its single-phase bacterium mutation detection of Salmonella1,4,
[5], the detection primer group of 12:i:- parting detection, is made of following primer:
For the site STMV1:
STMV1-F:5 '-GCAAAAGCAGGCTGAAG-3 ' (as shown in SEQ ID NO.8), STMV1-R:5 '-
GGCGCATTCTTACCCGAG-3 ' (as shown in SEQ ID NO.9)；
For the site STMV2:
STMV2-F:5 '-CGTGATGCTCTGATTTTGCT-3 ' (as shown in SEQ ID NO.10), STMV2-R:5 '-
CCGTAAGCGGGAATCGTA-3 ' (as shown in SEQ ID NO.11).
Third object of the present invention is to provide one kind to be used for Salmonella typhimurtum or its single-phase bacterium mutation detection of Salmonella1,4,
[5], the site VNTR of 12:i:- parting detection, the described site the VNTR number is STMV1, STMV2, STTR9, STTR5,
STTR6, STTR10 and STTR3；
The nucleotide sequence of the STMV1 is as shown in SEQ ID NO.1；
The nucleotide sequence of the STMV2 is as shown in SEQ ID NO.2；
The nucleotide sequence of the STTR9 is as shown in SEQ ID NO.3；
The nucleotide sequence of the STTR5 is as shown in SEQ ID NO.4；
The nucleotide sequence of the STTR6 is as shown in SEQ ID NO.5；
The nucleotide sequence of the STTR10 is as shown in SEQ ID NO.6；
The nucleotide sequence of the STTR3 is as shown in SEQ ID NO.7.
Fourth object of the present invention is to provide a kind of for Salmonella typhimurtum or its single-phase bacterium mutation detection of Salmonella1,4,
[5], the detection primer group of 12:i:- parting detection, is made of following primer:
For the site STMV1:
STMV1-F:5 '-GCAAAAGCAGGCTGAAG-3 ' (as shown in SEQ ID NO.8), STMV1-R:5 '-
GGCGCATTCTTACCCGAG-3 ' (as shown in SEQ ID NO.9)；
For the site STMV2:
STMV2-F:5 '-CGTGATGCTCTGATTTTGCT-3 ' (as shown in SEQ ID NO.10), STMV2-R:5 '-
CCGTAAGCGGGAATCGTA-3 ' (as shown in SEQ ID NO.11)；
For the site STTR9:
STTR9-F:5 '-AGAGGCGCTGCGATTGACGATA-3 ' (as shown in SEQ ID NO.12), STTR9-R:5 '-
CATTTTCCACAGCGGCAGTTTTTC-3 ' (as shown in SEQ ID NO.13)；
For the site STTR5:
STTR5-F:5 '-ATGGCGAGGCGAGCAGCAGT-3 ' (as shown in SEQ ID NO.14), STTR5-R:5 '-
GGTCAGGCCGAATAGCAGGAT-3 ' (as shown in SEQ ID NO.15)；
For the site STTR6:
STTR6-F:5 '-TCGGGCATGCGTTGAAA-3 ' (as shown in SEQ ID NO.16), STTR6-R:5 '-
CTGGTGGGGAGAATGACTGG-3 ' (as shown in SEQ ID NO.17)；
For the site STTR10:
STTR10-F:5 '-CGGGCGCGGCTGGAGTATTTG-3 ' (as shown in SEQ ID NO.18), STTR10-R:5 '-
GAAGGGGCCGGGCAGAGACAGC-3 ' (as shown in SEQ ID NO.19)；
For the site STTR3:
STTR3-F:5 '-CCCCCTAAGCCCGATAATGG-3 ' (as shown in SEQ ID NO.20), STTR3-R:5 '-
TGACGCCGTTGCTGAAGGTAATAA-3 ' (as shown in SEQ ID NO.21).
Fifth object of the present invention is to provide a kind of Salmonella typhimurtum or its single-phase bacterium mutation detection of Salmonella1,4,[5],
12:i:- parting detecting reagent, including PCR amplification reagent, the detection primer group.
Sixth object of the present invention is to provide a kind of Salmonella typhimurtum of the diagnosing and treating purpose of non-disease or its
Single-phase bacterium mutation detection of Salmonella1, 4, [5], 12:i:- parting determination method, comprising the following steps: it is husky to extract mouse typhus to be measured
The genomic DNA of door bacterium is as template, respectively using the STMV1-F/STMV1-R and STMV2-F/STMV2-R as amplification
Primer, or respectively with described STMV1-F/STMV1-R, STMV2-F/STMV2-R, STTR9-F/STTR9-R, STTR5-F/
STTR5-R, STTR6-F/STTR6-R, STTR10-F/STTR10-R and STTR3-F/STTR3-R are carried out as amplimer
Amplified production is sequenced in PCR amplification, carries out parting to Salmonella typhimurtum to be measured according to sequencing result.
The reaction system of the PCR amplification is preferably 25 μ L, including 10 × PCR buffer 2.5 μ L, 3.0mmol/L
MgCl2, 250 μm of ol/L dNTP, each 120nmol/L of upstream and downstream primer, 1.5U Taq enzyme, template 2 μ L, surplus ddH2O。
The amplification condition of the PCR amplification is preferably 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 45s, 58 DEG C of annealing 45s,
72 DEG C of extension 45s carry out 30 circulations altogether；72 DEG C of extension 10min.
7th purpose of the invention is to provide the detection primer group in Salmonella typhimurtum or its single-phase bacterium mutation
Detection of Salmonella1, 4, [5], 12:i:- parting detection in application.
The present invention is directed to Salmonella typhimurtum and its single-phase bacterium mutation detection of Salmonella1, 4, [5], the VNTR that 12:i:- is newly screened
Design specific nucle primer sequence in site (STMV1 and STMV2).And be combined for 7 site VNTR-STMV1, STMV2,
The primer sets of STTR9, STTR5, STTR6, STTR10 and STTR3 establish the site MLVA-2 method (detection STMV1 and STMV2 respectively
Site) and the site MLVA-7 method (the detection site STMV1, STMV2, STTR9, STTR5, STTR6, STTR10 and STTR3).By
A large amount of Salmonella typhimurtums and its single-phase bacterium mutation detection of Salmonella1, 4, [5], the detection verifying of 12:i:- bacterial strain, detection of the invention point
Analysis method can be directly to Salmonella typhimurtum and its single-phase bacterium mutation detection of Salmonella1, 4, [5], 12:i:- carries out stable parting inspection
It surveys, only analyzes 2 genes, parting efficiency is suitable with traditional MLST classifying method, only analyzes 7 genes, and parting efficiency is higher than
It is combined original site MLST and MLVA-5 classifying method.
Detailed description of the invention:
Fig. 1 is Salmonella typhimurtum and its serovar detection of Salmonella1, 4, [5], the MLST clustering of 12:i:-
(Achtman et al.,2012,PLoS Pathog)；Caption: each circle represents a kind of ST type, is divided in circle with sector
It cuts and represents one plant of bacterium, the number beside circle represents ST type.The connected main affiliation represented between different ST types of straight line, tail
The name of the cross that portion has the straight line of strigula to represent same level, eBG marks in circular white frame.
Specific embodiment:
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1:
1, the design of specific primer sequence:
Known Salmonella typhimurtum reference culture LT2 sequence information is analyzed, 2 specific positions VNTR are newly screened
Point --- STMV1 and STMV2 (table 1), separately designs specific nucle primer sequence.
1) Salmonella typhimurtum and its single-phase bacterium mutation detection of Salmonella are directed to1, 4,2 newly screened of [5], 12:i:- are special
The property site VNTR --- STMV1 and STMV2 and 5 known site VNTR, the different sites VNTR is shown in Table 1.
Position (nt) of the site table 1.VNTR in reference culture Salmonella Typhi LT2
| Site | Position (nt) of the site VNTR in reference culture Salmonella Typhi LT2 |
| STMV1 | 814967-815497 |
| STMV2 | 1958188-1958366 |
| STTR9 | 3246601-3246781 |
| STTR5 | 3184509-3184767 |
| STTR6 | 2730724-2731065 |
| STTR10 | 53512-53883 |
| STTR3 | 3629466-3629955 |
2) Salmonella typhimurtum and its single-phase bacterium mutation detection of Salmonella are directed to1, 4,2 newly screened of [5], 12:i:- are special
The primer in the property site VNTR --- STMV1 and STMV2,2 pairs of primers of design and 5 known sites VNTR, different VNTR
The primer in site is shown in Table 2.
The site table 2.VNTR amplimer
By taking Salmonella typhimurtum reference culture LT2 as an example, each site VNTR primer and extension increasing sequence:
1) (as shown in SEQ ID NO.1, underscore partial sequence is respectively by STMV1 primer and extension increasing sequence 531bp
The complementary base sequences thereof of STMV1-F sequence and STMV1-R, overstriking region are flanking sequence, and the centre in overstriking region is to repeat list
Position is also referred to as core sequence)
2) (as shown in SEQ ID NO.2, underscore partial sequence is respectively by STMV2 primer and extension increasing sequence 179bp
The complementary base sequences thereof of STMV2-F sequence and STMV2-R, overstriking region are flanking sequence, and the centre in overstriking region is to repeat list
Position is also referred to as core sequence)
3) (as shown in SEQ ID NO.3, underscore partial sequence is respectively by STTR9 primer and extension increasing sequence 180bp
The complementary base sequences thereof of STTR9-F sequence and STTR9-R, overstriking region are flanking sequence, and the centre in overstriking region is to repeat list
Position is also referred to as core sequence)
4) (as shown in SEQ ID NO.4, underscore partial sequence is respectively by STTR5 primer and extension increasing sequence 259bp
The complementary base sequences thereof of STTR5-F sequence and STTR5-R, overstriking region are flanking sequence, and the centre in overstriking region is to repeat list
Position is also referred to as core sequence)
5) (as shown in SEQ ID NO.5, underscore partial sequence is respectively by STTR6 primer and extension increasing sequence 342bp
The complementary base sequences thereof of STTR6-F sequence and STTR6-R, overstriking region are flanking sequence, and the centre in overstriking region is to repeat list
Position is also referred to as core sequence)
6) (as shown in SEQ ID NO.6, underscore partial sequence is respectively by STTR10 primer and extension increasing sequence 371bp
The complementary base sequences thereof of STTR10-F sequence and STTR10-R, overstriking region are flanking sequence, and the centre in overstriking region is to repeat
Unit is also referred to as core sequence)
7) (as shown in SEQ ID NO.7, underscore partial sequence is respectively by STTR3 primer and extension increasing sequence 490bp
The complementary base sequences thereof of STTR3-F sequence and STTR3-R, overstriking region are flanking sequence, and the centre in overstriking region is to repeat list
Position is also referred to as core sequence)
2, using the application in above-mentioned site and primer pair Salmonella typhimurtum MLVA Genotyping, specific steps:
(1) DNA of bacteria extracts
Own method can be used or commercial DNA of bacteria extracts kit extracts the DNA of surveyed bacterial strain.
(2) PCR reaction system and amplification condition
PCR reaction system: 10 × PCR buffer, 2.5 μ L, 3.0mmol/L MgCl2, 250 μm of ol/L dNTP, up and down
Swim each 120nmol/L of primer, 1.5U Taq enzyme, 2 μ L of template, distilled water polishing to 25 μ L of total volume.
PCR amplification condition: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 45s, 58 DEG C of annealing 45s, 72 DEG C of extension 45s are carried out altogether
30 circulations；72 DEG C of extension 10min.Positive control (Salmonella typhimurtum reference culture LT2 genomic DNA) and sky are set up simultaneously
White control (ddH2O)。
(3) sequencing analysis
Amplification sample is subjected to sequencing analysis, obtains the sequence information in the site target VNTR.
(4) result judgement is carried out referring to following formula:
According to sequencing result, number (Repeart number)={ primer size (Length of of repetitive sequence
Product the clip size (Flanking size) around)-repetitive sequence }/each repetitive sequence size (Length of
each repeat)。
(5) result indicates
According to the number of each site repetitive sequence in each site pcr amplified fragment sequencing result, the site MLVA-2 method
(only detect this 2 sites STMV1 and STMV2) result indicates with n-n, the site MLVA-7 method (detection STMV1, STMV2,
This 7 sites STTR9, STTR5, STTR6, STTR10 and STTR3) result indicates with n-n-n-n-n-n-n；Bacterial strain 1 is ST19
7 site VNTR STMV1, STMV2, STTR9, STTR5, STTR6, STTR10 and STTR3 specificity of type Salmonella typhimurtum
Primer PCR reaction product sequence verification is as a result, its repetitive sequence number is respectively STMV1: repetitive sequence number Rep=9；STMV2:
Repetitive sequence number Rep=3；STTR9: repetitive sequence number Rep=4；STTR5: repetitive sequence number Rep=13；STTR6: sequence is repeated
Columns Rep=13；STTR10: repetitive sequence number Rep=10；STTR3: repetitive sequence number Rep=2/11；The MLVA-2 of bacterial strain 1
Site method MLVA type is expressed as 9-3, and the site the MLVA-7 method MLVA type of bacterial strain 1 is expressed as 9-3-4-13-13-10-211.
Bacterial strain 2 is 7 site VNTR STMV1, STMV2 of ST34 type Salmonella typhimurtum, STTR9, STTR5, STTR6,
STTR10 and STTR3 specific primer PCR reaction product sequence verification is as a result, its repetitive sequence number is respectively STMV1: repeating
Sequence number Rep=9；STMV2: repetitive sequence number Rep=4；STTR9: repetitive sequence number Rep=3；STTR5: repetitive sequence number
Rep=11；STTR6: repetitive sequence number Rep=9；STTR10: repetitive sequence number Rep=8；STTR3: repetitive sequence number Rep=
2/11；The site the MLVA-2 method MLVA type of bacterial strain 2 is expressed as 9-4, and the site the MLVA-7 method MLVA type of bacterial strain 2 is expressed as 9-
4-3-11-9-8-211。
Bacterial strain 3 is 7 site VNTR STMV1, STMV2 of ST36 type Salmonella typhimurtum, STTR9, STTR5, STTR6,
STTR10 and STTR3 specific primer PCR reaction product sequence verification is as a result, its repetitive sequence number is respectively STMV1: repeating
Sequence number Rep=8；STMV2: repetitive sequence number Rep=3；STTR9: repetitive sequence number Rep=2；STTR5: repetitive sequence number
Rep=12；STTR6: repetitive sequence number Rep=6；STTR10: repetitive sequence number Rep=10；STTR3: repetitive sequence number Rep=
3/11；The site the MLVA-2 method MLVA type of bacterial strain 3 is expressed as 8-3, and the site the MLVA-7 method MLVA type of bacterial strain 3 is expressed as 8-
3-2-12-6-10-311。
46 plants of Salmonella typhimurtums of laboratory preservation are analyzed, 46 plants of bacterium are divided into 22 plants of ST19,19 plants by MLST
ST34 and 5 plant of ST36.The genotyping result of MLVA is shown in Table all 9 Hes of repeat number in 3,22 plants of sites ST19 type bacterial strain STMV1
The repeat number in the site STMV2 all 3, the repeat number all 9 in the site STMV1 of 19 plants of ST34 type bacterial strains and the site STMV2
Repeat number all 4, the repeat number in the site STMV1 of 5 plants of ST36 type bacterial strains is 8 and the repeat number in the site STMV2 is 3.
The site MLVA-2 method only analyzes 2 genes, and parting efficiency is suitable with traditional MLST classifying method, and the site MLVA-7 method is only analyzed
7 genes, greatly simplifie operating procedure, improve parting efficiency, save a large amount of human and material resources and financial resources etc..
3. 46 plants of Salmonella typhimurtum strain typing results of table
* note: " NA " representative does not detect the site in table
The sequencing initial data of 150 plants of Salmonella typhimurtum correlation ST type bacterial strains has been downloaded from NCBI-ENA database,
Spliced and assembled, MLST analysis is carried out to the strain gene group that success assembles, confirms bacterial strain ST type, and carry out to bacterial strain
The analysis in the site STMV1 and STMV2.60 plants of Salmonella typhimurtum correlation ST type bacterium have been downloaded from NCBI-Genome database
The genome of strain completes graphic sequence, analyzes bacterial strain ST type, and the analysis in the site STMV1 and STMV2 is carried out to bacterial strain.Finally obtain
Obtained STMV1 the and STMV2 Locus Analysis in Shoots result (being shown in Table 4) of 80 plants of bacterial strains.The repetition in the site STMV1 of 25 plants of ST36 type bacterial strains
The repeat number all 9 in all sites 8,37 plants of ST19 type bacterial strain STMV1 of number and the repeat number all 3,5 in the site STMV2
The repeat number all 9 in the site STMV1 of strain ST34 type bacterial strain and the repeat number all 4 in the site STMV2, other ST bacterial strains are detailed
It is shown in Table 4.
4. 80 plants of Salmonella typhimurtum bacterial strain databases of table download genome STMV1 and STMV2 Locus Analysis in Shoots result
3, above-mentioned site and the single-phase bacterium mutation detection of Salmonella of primer pair mouse typhus are utilized1, 4, [5], 12:i:-MLVA gene point
The application of type, specific steps:
(1) DNA of bacteria extracts
Own method can be used or commercial DNA of bacteria extracts kit extracts the DNA of surveyed bacterial strain.
(2) PCR reaction system and amplification condition
PCR reaction system: 10 × PCR buffer, 2.5 μ L, 3.0mmol/L MgCl2, 250 μm of ol/L dNTP, up and down
Swim each 120nmol/L of primer, 1.5U Taq enzyme, 2 μ L of template, distilled water polishing to 25 μ L of total volume.
PCR amplification condition: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 45s, 58 DEG C of annealing 45s, 72 DEG C of extension 45s are carried out altogether
30 circulations；72 DEG C of extension 10min.Positive control (Salmonella typhimurtum reference culture LT2 genomic DNA) and sky are set up simultaneously
White control (ddH2O)。
(3) sequencing analysis
Amplification sample is subjected to sequencing analysis, obtains the sequence information in the site target VNTR.
(4) result judgement is carried out referring to following formula:
According to sequencing result, number (Repeart number)={ primer size (Length of of repetitive sequence
Product the clip size (Flanking size) around)-repetitive sequence }/each repetitive sequence size (Length of
each repeat)。
(5) result indicates
According to the number of each site repetitive sequence in each site pcr amplified fragment sequencing result, the site MLVA-2 method
(only detect this 2 sites STMV1 and STMV2) result indicates with n-n, the site MLVA-7 method (detection STMV1, STMV2,
This 7 sites STTR9, STTR5, STTR6, STTR10 and STTR3) result indicates with n-n-n-n-n-n-n；Bacterial strain 4 is ST19
The single-phase bacterium mutation detection of Salmonella of type mouse typhus1, 4, [5], 7 site VNTR STMV1, STMV2 of 12:i:-, STTR9, STTR5,
STTR6, STTR10 and STTR3 specific primer PCR reaction product sequence verification are as a result, its repetitive sequence number is respectively
STMV1: repetitive sequence number Rep=9；STMV2: repetitive sequence number Rep=3；STTR9: repetitive sequence number Rep=2；STTR5: weight
Complex sequences number Rep=10；STTR6: repetitive sequence number Rep=5；STTR10: repetitive sequence number Rep=NA；STTR3: sequence is repeated
Columns Rep=2/12；The site the MLVA-2 method MLVA type of bacterial strain 4 is expressed as 9-3, the site the MLVA-7 method MLVA type of bacterial strain 4
9-3-2-10-5-NA-212 is not expressed as it.
Bacterial strain 5 is the single-phase bacterium mutation detection of Salmonella of ST34 type mouse typhus1, 4, [5], 7 site VNTR STMV1 of 12:i:-,
STMV2, STTR9, STTR5, STTR6, STTR10 and STTR3 specific primer PCR reaction product sequence verification are as a result, it is repeated
Sequence number is respectively STMV1: repetitive sequence number Rep=9；STMV2: repetitive sequence number Rep=4；STTR9: repetitive sequence number
Rep=3；STTR5: repetitive sequence number Rep=13；STTR6: repetitive sequence number Rep=5；STTR10: repetitive sequence number Rep=
NA；STTR3: repetitive sequence number Rep=2/11；The site the MLVA-2 method MLVA type of bacterial strain 5 is expressed as 9-4, bacterial strain 5
The site MLVA-7 method MLVA type is expressed as 9-4-3-13-5-NA-211.
To the single-phase bacterium mutation detection of Salmonella of 13 plants of mouse typhus of laboratory preservation1, 4, [5], 12:i:- is analyzed, 13 plants of mouse
The single-phase bacterium mutation detection of Salmonella of typhoid fever1, 4, [5], all ST34 types of 12:i:-.MLVA genotyping result is shown in Table 5,13 plants of ST34 type bacterium
The repeat number all 9 in the site STMV1 of strain and the repeat number all 4 in the site STMV2.
The single-phase bacterium mutation detection of Salmonella of 5. 13 plants of mouse typhus of table1, 4, [5], 12:i:- strain typing result
| Bacterial strain | ST(MLST) | STMV1 | STMV2 | STTR9 | STTR5 | STTR6 | STTR10 | STTR3 |
| 1 | 34 | 9 | 4 | 3 | 10 | 9 | NA | 2/11 |
| 2 | 34 | 9 | 4 | 3 | 14 | 12 | NA | 2/11 |
| 3 | 34 | 9 | 4 | 3 | 13 | 9 | 13 | 2/11 |
| 4 | 34 | 9 | 4 | 3 | 13 | 9 | 6 | 2/11 |
| 5 | 34 | 9 | 4 | 3 | 11 | 9 | NA | 2/11 |
| 6 | 34 | 9 | 4 | 3 | 12 | 9 | NA | 2/11 |
| 7 | 34 | 9 | 4 | 3 | 5 | 9 | NA | 2/11 |
| 8 | 34 | 9 | 4 | 3 | 14 | 9 | 11 | 2/11 |
| 9 | 34 | 9 | 4 | 3 | 13 | 13 | NA | 2/11 |
| 10 | 34 | 9 | 4 | 3 | 13 | 9 | 11 | 2/11 |
| 11 | 34 | 9 | 4 | 3 | 13 | 9 | 10 | 2/11 |
| 12 | 34 | 9 | 4 | 3 | 13 | 13 | NA | 2/11 |
| 13 | 34 | 9 | 4 | 3 | 14 | 11 | NA | 2/11 |
The single-phase bacterium mutation detection of Salmonella of 57 plants of mouse typhus has been downloaded from NCBI-Genome database1, 4, [5], 12:i:- correlation
The genome sequence of ST type bacterial strain analyzes the ST type of bacterial strain, and the analysis in the site STMV1 and STMV2 is carried out to bacterial strain.It obtains
Obtained STMV1 and STMV2 Locus Analysis in Shoots (site MLVA-2 method) result (being shown in Table 6) of 57 plants of bacterial strains.8 plants of ST19 type bacterial strains
The repeat number in the site STMV1 all 9 and the repeat number in the site STMV2 all 3, the site STMV1 of 49 plants of ST34 type bacterial strains
Repeat number all 9 and the site STMV2 repeat number all 4.
The single-phase bacterium mutation detection of Salmonella of 6. mouse typhus of table1, 4, [5], 12:i:- bacterial strain database downloads genome MLVA-2
Point method analyzes result
Sequence table
<110>Guangdong Microbes Inst (microbiological analysis inspection center, Guangdong Province)
<120>for Salmonella typhimurtum or the site VNTR, detection primer group and the detection point of its single-phase bacterium mutation parting detection
Analysis method
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 531
<212> DNA
<213>Salmonella Typhi LT2 (Salmonella typhimurium LT2)
<400> 1
gcaaaagcag gctgaagaag cggcaaaact ggcgcaacag cagcagcaac aggccgaaga 60
agcggcgaaa gcggcggcgg acgcgaagaa aaaagcggag gccgaggcgg cgaaagcggc 120
ggcggacgcg aagaagaaag cggaagccga agcggtaaaa gcggcagcgg acgcgaagaa 180
gaaagcggaa gccgaagcgg cgaaagcggc ggcggacgcg aagaagaaag cagaggccga 240
agcggcgaaa gcggcggcgg aggcgaagaa gaaagcggaa gccgaagcgg cgaaagcggc 300
ggcggaggcg aagaagaaag cggatgccga ggcggcgaaa gcggcggcgg aggcgaagaa 360
gaaagcggat gccgcggcgg cgaaagcggc ggcggaggcg aagaagaaag cggatgccgc 420
ggcggcgaaa gcagcggcgg acgctaagaa gaaagcggct gccgaaaaag cggccgccgc 480
agaaggcgtc gacgatctgc ttggcgatct cagctcgggt aagaatgcgc c 531
<210> 2
<211> 179
<212> DNA
<213>Salmonella Typhi LT2 (Salmonella typhimurium LT2)
<400> 2
cgtgatgctc tgattttgct gtaaaagaaa tgttaaactg gatttggatc tgatataacg 60
aaaaagcccg gaatacagaa attctggaaa atttcggaaa tttcggaaat ttcggattat 120
tgatcataag caactgattt ataagaaaaa aagagaccga atacgattcc cgcttacgg 179
<210> 3
<211> 180
<212> DNA
<213>Salmonella Typhi LT2 (Salmonella typhimurium LT2)
<400> 3
agaggcgctg cgattgacga tatctatgac tttatggaat agcatattca ctatgtgggt 60
tgcggaggca tcgcgtcgtt tgcgatgtct gcgatgtctg cgatgtctgc gatgtctgcg 120
gtggatatcg ccctttggga tttgaaaggc aaacgtgaaa aactgccgct gtggaaaatg 180
<210> 4
<211> 259
<212> DNA
<213>Salmonella Typhi LT2 (Salmonella typhimurium LT2)
<400> 4
atggcgaggc gagcagcagt ggctggcggg aaaccaccat cacgaccacg accacgacca 60
cgaccacgac cacgaccacg accacgacca cgaccacgac cacgaccacg accacgacca 120
tcatggtcac atacatccgg aaggtgcaac gtcaaaagcg tatcaggatg cccatgaacg 180
cgcccatgct gccgatattc aacgccgttt tgatggtcaa acagtgacta atggacagat 240
cctgctattc ggcctgacc 259
<210> 5
<211> 342
<212> DNA
<213>Salmonella Typhi LT2 (Salmonella typhimurium LT2)
<400> 5
tcgggcatgc gttgaaaacc tgcaaaaaaa gaaaaatatc gatgcttatt attttttctt 60
taattaaatt ttcgctcaac aaacttaatt attaatccaa tgacagtgaa gtgtgaacta 120
tgctgagcat aaacgacatc aatagcaagg gcaagggcaa gggcaagggc aagggcaagg 180
gcaagggcaa gggcaagggc aagggcaagg gcaagggcaa gggcaatctg agtatttaca 240
ggtgaaatta tgaaaaatag tatattttta ccattgcttc tggtgctgtc ggcaacgacc 300
ttttcggcat tggtgatggc tgccagtcat tctccccacc ag 342
<210> 6
<211> 371
<212> DNA
<213>Salmonella Typhi LT2 (Salmonella typhimurium LT2)
<400> 6
cgggcgcggc tggagtattt gcgcaactcc cggacaagaa tgccccggaa tatgccagaa 60
aagaggatga ggttaaaaaa tagcgccatt cctgatgcat tctgcctgtc gctggccgga 120
ttttacgcat gccgggtgat gtttttttct ggccggggtg tgtgattttc ttcctgccgg 180
tgataatacg ctgcctgttc ctgttcctgt tcctgttcct gttcctgttc ctgttcctgt 240
tcctgttcct gttcattctg ctgctgatat tccctgacag atttcgctga cgccgggcga 300
cttccctgac gggaaccggg actgtcatgc tgcgcaccga gccagctgcg ctgtctctgc 360
ccggcccctt c 371
<210> 7
<211> 490
<212> DNA
<213>Salmonella Typhi LT2 (Salmonella typhimurium LT2)
<400> 7
ccccctaagc ccgataatgg cggcgacgtc accccgcccg acgatggcgg caacgtcacc 60
ccgcccgacg atggcggcaa cgtcaccccg cccgacgatg gcggcgatga caatgtgacc 120
ccgcccgacg atagtggcga tgacgatgtg gccccgcctg acgatagcgg cgatgacgat 180
gtaaccccgc ccgacgatag cggcgatgat gatgtgaccc cgcccgacga tagcggcgat 240
ggcgatgtga ccccgcccga cgatagcggc gatgacgatg taaccccgcc cgacgatagc 300
ggcgatgacg atgtgacccc gcctgacgat agcggcgatg acgatgtaac cccgcccgac 360
gatagcggcg atgacgatgt gaccccgccc gatgatagcg gcgatgacga tgtaaccccg 420
cccgatgata gcggcgatga cgacgacacg cccccagatg actctgttat taccttcagc 480
aacggcgtca 490
<210> 8
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gcaaaagcag gctgaag 17
<210> 9
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ggcgcattct tacccgag 18
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
cgtgatgctc tgattttgct 20
<210> 11
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ccgtaagcgg gaatcgta 18
<210> 12
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
agaggcgctg cgattgacga ta 22
<210> 13
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
cattttccac agcggcagtt tttc 24
<210> 14
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
atggcgaggc gagcagcagt 20
<210> 15
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ggtcaggccg aatagcagga t 21
<210> 16
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
tcgggcatgc gttgaaa 17
<210> 17
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ctggtgggga gaatgactgg 20
<210> 18
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
cgggcgcggc tggagtattt g 21
<210> 19
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gaaggggccg ggcagagaca gc 22
<210> 20
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ccccctaagc ccgataatgg 20
<210> 21
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
tgacgccgtt gctgaaggta ataa 24
Claims (9)
1. one kind is used for Salmonella typhimurtum or its single-phase bacterium mutation detection of Salmonella 1,4, [5], the position VNTR of 12:i:- parting detection
Point, which is characterized in that the site the VNTR number is STMV1 and STMV2；
The nucleotide sequence of the STMV1 is as shown in SEQ ID NO.1；
The nucleotide sequence of the STMV2 is as shown in SEQ ID NO.2.
2. one kind is used for Salmonella typhimurtum or its single-phase bacterium mutation detection of Salmonella 1,4, the detection of [5], the detection of 12:i:- parting is drawn
Object group, which is characterized in that be made of following primer:
For the site STMV1:
STMV1-F:5 '-GCAAAAGCAGGCTGAAG-3 ', STMV1-R:5 '-GGCGCATTCTTACCCGAG-3 '；
For the site STMV2:
STMV2-F:5 '-CGTGATGCTCTGATTTTGCT-3 ', STMV2-R:5 '-CCGTAAGCGGGAATCGTA-3 '.
3. one kind is used for Salmonella typhimurtum or its single-phase bacterium mutation detection of Salmonella 1,4, [5], the position VNTR of 12:i:- parting detection
Point, which is characterized in that the described site the VNTR number be STMV1, STMV2, STTR9, STTR5, STTR6, STTR10 and
STTR3；
The nucleotide sequence of the STMV1 is as shown in SEQ ID NO.1；
The nucleotide sequence of the STMV2 is as shown in SEQ ID NO.2；
The nucleotide sequence of the STTR9 is as shown in SEQ ID NO.3；
The nucleotide sequence of the STTR5 is as shown in SEQ ID NO.4；
The nucleotide sequence of the STTR6 is as shown in SEQ ID NO.5；
The nucleotide sequence of the STTR10 is as shown in SEQ ID NO.6；
The nucleotide sequence of the STTR3 is as shown in SEQ ID NO.7.
4. one kind is used for Salmonella typhimurtum or its single-phase bacterium mutation detection of Salmonella 1,4, the detection of [5], the detection of 12:i:- parting is drawn
Object group, which is characterized in that be made of following primer:
For the site STMV1:
STMV1-F:5 '-GCAAAAGCAGGCTGAAG-3 ', STMV1-R:5 '-GGCGCATTCTTACCCGAG-3 '；
For the site STMV2:
STMV2-F:5 '-CGTGATGCTCTGATTTTGCT-3 ', STMV2-R:5 '-CCGTAAGCGGGAATCGTA-3 '；
For the site STTR9:
STTR9-F:5 '-AGAGGCGCTGCGATTGACGATA-3 ', STTR9-R:5 '-CATTTTCCACAGCGGCAGTTTTTC-
3'；
For the site STTR5:
STTR5-F:5 '-ATGGCGAGGCGAGCAGCAGT-3 ', STTR5-R:5 '-GGTCAGGCCGAATAGCAGGAT-3 '；
For the site STTR6:
STTR6-F:5 '-TCGGGCATGCGTTGAAA-3 ', STTR6-R:5 '-CTGGTGGGGAGAATGACTGG-3 '；
For the site STTR10:
STTR10-F:5 '-CGGGCGCGGCTGGAGTATTTG-3 ', STTR10-R:5 '-GAAGGGGCCGGGCAGAGACAGC-
3'；
For the site STTR3:
STTR3-F:5 '-CCCCCTAAGCCCGATAATGG-3 ', STTR3-R:5 '-TGACGCCGTTGCTGAAGGTAATAA-
3’。
5. a kind of Salmonella typhimurtum or its single-phase bacterium mutation detection of Salmonella Isosorbide-5-Nitrae, [5], 12:i:- parting detecting reagent, feature
It is, including detection primer group described in PCR amplification reagent, claim 2 or 4.
6. a kind of Salmonella typhimurtum of the diagnosing and treating purpose of non-disease or its single-phase bacterium mutation detection of Salmonella 1,4, [5], 12:
I:- parting determination method, which comprises the following steps: the genomic DNA for extracting Salmonella typhimurtum to be measured is made
For template, respectively using STMV1-F/STMV1-R as claimed in claim 2 and STMV2-F/STMV2-R as amplimer, or
Respectively with STMV1-F/STMV1-R, STMV2-F/STMV2-R, STTR9-F/STTR9-R, STTR5-F/ as claimed in claim 4
STTR5-R, STTR6-F/STTR6-R, STTR10-F/STTR10-R and STTR3-F/STTR3-R are carried out as amplimer
Amplified production is sequenced in PCR amplification, carries out parting to Salmonella typhimurtum to be measured according to sequencing result.
7. Salmonella typhimurtum according to claim 6 or its single-phase bacterium mutation detection of Salmonella 1,4, [5], the inspection of 12:i:- parting
Survey analysis method, which is characterized in that the reaction system of the PCR amplification is 25 μ L, including 10 × PCR buffer, 2.5 μ L,
3.0mmol/L MgCl2, 250 μm of ol/L dNTP, each 120nmol/L of upstream and downstream primer, 1.5U Taq enzyme, 2 μ L of template, surplus
For ddH2O。
8. Salmonella typhimurtum according to claim 6 or its single-phase bacterium mutation detection of Salmonella 1,4, [5], the inspection of 12:i:- parting
Survey analysis method, which is characterized in that the amplification condition of the PCR amplification is 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 45s, 58
DEG C annealing 45s, 72 DEG C of extensions 45s, altogether carry out 30 recycle；72 DEG C of extension 10min.
9. detection primer group described in claim 2 or 4 is in Salmonella typhimurtum or its single-phase bacterium mutation detection of Salmonella 1,4, [5],
Application in the detection of 12:i:- parting.
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| CN111020041B (en) * | 2019-12-30 | 2022-10-11 | 广东省微生物研究所（广东省微生物分析检测中心） | 16 different serotype salmonella specific new molecular targets and rapid detection method thereof |
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