CN105296635A - PCR-HRM primer for quickly detecting salmonella pullorum and application thereof - Google Patents
PCR-HRM primer for quickly detecting salmonella pullorum and application thereof Download PDFInfo
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Abstract
The invention discloses a PCR-HRM primer for quickly detecting salmonella pullorum and application thereof. The PCR-HRM primer for quickly detecting salmonella pullorum and the application are established for the first time. A method has the advantages of being simple to operate, quick, high in specificity, high in sensitivity and throughput and low in cost. The PCR-HRM primer can be widely applied to veterinarian clinical detection, and the quick and efficient method is provided for purification of salmonella pullorum in breeding plants. During the detection, only saturated fluorescent dye needs to be added into a PCR; the clinical detection process is simplified, the detection time is greatly shortened, and the time is saved by 2/3 compared with a conventional national standard identification method. The salmonella pullorum can be distinguished well, and the problem that a conventional detection method is low in salmonella pullorum identification accuracy is solved. The PCR-HRM primer is high in sensitivity, and the lowest detection limit can be 34 copy numbers per microliter. The PCR-HRM primer has extensive application prospects in rapid diagnosis of salmonella pullorum.
Description
Technical field
The present invention relates to biotechnology and medical field, especially microorganism detection, molecular diagnosis.Be specifically related to a kind of PCR-HRM primer and application thereof of rapid detection S. pullonum.
Background technology
S. pullonum (SalmonellaPullorum) belongs to intestinal bacilli section salmonella, a member in D serotype.Main infection chicken and turkey, clinical table performance draws white paste loose stool for feature with chick, and mortality ratio is high.Rettger is Salmonella pullorum disease in reported first in 1899, next year by this sick called after " chick lethality septicemia " nineteen twenty-nine formally by this sick called after " white dysentery ".All have generation and the existence of this disease in the area of all raising chickens and turkey, in Mammals, rabbit has high susceptibility, has people once in pig body, to be separated to this bacterium, and children also occasionally have the report infecting this disease.At present, one of main source of mankind's Salmonella infection and food poisoning has been become by salmonella-polluted poultry and related products.In addition, along with the fast development of aviculture, Salmonella pullorum disease has become one of poultry bacteriosis the most always, and be listed as the bacteriosis of national regulation purification, the financial loss caused every year is considerable.
Quick discriminating and diagnosis S. pullonum are the bases of purification and control, traditional authentication method cycle is long, sensitivity is low, poor specificity, the pre-liquid BPW that increases is needed to increase bacterium (8 ~ 18h) as gathered sample, then selective enrichment broth (TTB, SC etc.) cultivates (18 ~ 24h), again enrichment liquid is inoculated into the dull and stereotyped upper cultivation (18 ~ 24h) of colour developing, then selects suspicious bacterium colony and carry out biochemical identification and serological identification (12 ~ 48h).Serodiagnosis and avian infectious bronchitis nephritis virus (SalmonellaGallinarum) is utilized to have very high cross agglutination.In addition, Salmonella enteritidis (9,12:g, m:-) unpowered mutation (9,12:-:-), because losing H phase (g, m) antigen presentation ability and (g and the m factor) cannot be confirmed with diagnostic serum, and it is pure in antigen formula (9,12:-:-) then S. pullonum can be mistaken for, so advocate the background of purifying aquaculture field Salmonellas in country under, find a kind of efficient, special, fast S. pullonum detection method there is important public health meaning and economic benefit.
High resolving power melting curve (high-resolutionmelting, HRM) be a kind of genetic analysis new technology forming different shape melting curve based on mononucleotide melting temperature (Tm) difference, there is high susceptibility, the difference of single base can be detected, the Tm value of double-stranded DNA can be made to change for SNP site base difference thus double-stranded DNA is successively untied in temperature-rise period, form different melt curve analysis shapes, the DNA molecular that fluorescence dye unwinds from local discharges, from fluorescence intensity and time curve, just can judge whether to there is SNP, and different SNP site, heterozygote and homozygote etc. all can affect the peak shape of melting curve, therefore HRM analyzes and can effectively distinguish different SNP site and different genotype.SYBR
tMgreenI has restraining effect to PCR, must use lower concentration; Even high density can not ditch in saturated insertion DNA double spirane structure, unsaturation state combines and dyestuff is reset in fusion processes, and dye molecule recombine, to the vacant site of double-stranded DNA, causes fluorescent signal not change, specificity decline.Saturable dye, also can not suppression PCR under saturation concentration (fluorescence is maximum); Ditch in the dyestuff saturated insertion DNA double spirane structure of high density, would not reset in DNA dehybridization procedure, melting curve has higher resolving power, and saturable dye mainly contains LCGreen, LCGreenPlus, CYTO9 etc.High resolving power melting curve cost in detection of pathogens is low, flux is high, speed is fast, result accurately, the limitation in not examined site, achieve real stopped pipe operation.Huge application prospect is had in detection of pathogens.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, primary and foremost purpose of the present invention is the PCR-HRM primer providing a kind of rapid detection S. pullonum.
Another object of the present invention is to a kind of PCR-HRM method that rapid detection S. pullonum is provided.The method has simple to operate, quick, high specificity, the highly sensitive and feature of high-throughput, low cost, can be widely used in veterinary clinic and detects, for the purification of cultivation factory S. pullonum provides method fast and efficiently.
Another object of the present invention is the application of the PCR-HRM primer providing described rapid detection S. pullonum.
Object of the present invention is achieved through the following technical solutions:
A PCR-HRM primer for rapid detection S. pullonum, its nucleotide sequence is as follows:
The complementary sequence of upstream primer rfbS-F:5 '-AAAGCAATATTCTTATGCCTA-3 ' or its Nucleotide;
Downstream primer rfbS-R:5 '-CACAATTTATGAATACTGCATC-3 ' or its nucleotide complementary sequence.
A PCR-HRM method for rapid detection S. pullonum, comprises the following steps:
A: extract DNA of bacteria;
B: be template with DNA, utilizes upstream primer rfbS-F described above, downstream primer rfbS-R and saturated fluorescence dyestuff to carry out amplified reaction, obtains amplified production;
C: carry out HRM analysis to amplified production, determines whether as S. pullonum.
Further, the pcr amplification reaction system described in step B is:
| PremixEx-Taq | 5.0μL |
| Primer rfbS-F | 0.5μL |
| Primer rfbS-R | 0.5μL |
| DNA profiling | 1.0μL |
| Saturated fluorescence dyestuff | 1.0μL |
| ddH 2O | 2.0μL |
| Total | 10μL |
Described saturated fluorescence dyestuff is preferably Evagreen dyestuff.
Described primer rfbS-F and the concentration of primer rfbS-R are respectively preferably 10nmol/ μ L;
Further, the amplified reaction program described in step B is:
Further, the concrete analysis process that HRM described in step C analyzes is: with the melting temperature (Tm) Tm value after the amplification of S. pullonum standard positive template for reference, and testing sample melting temperature (Tm) Tm value the value of the confidence (GCP) is greater than 95% for S. pullonum.
The PCR-HRM primer of described rapid detection S. pullonum can be applicable to the detection of S. pullonum, the purification of plant's S. pullonum, the risk assessment of S. pullonum in Molecule Epidemiology Investigation and poultry farming.
The present invention, relative to prior art, has following advantage and effect:
(1) the present invention establishes a kind of PCR-HRM primer of rapid detection S. pullonum first and applies, and it is easy to operate, only needs to add saturated fluorescence dyestuff during detection in PCR reaction; Simplify the flow process of Clinical Laboratory and substantially reduce detection time, the time saves 2/3 compared with traditional GB authentication method; And the method has the feature of high-throughput, low cost, once 384 samples can be checked at most.
(2) compared with normal PCR rear electrophoresis open operation, the present invention analyzes from PCR process to HRM and achieves real stopped pipe operation, and whole process PCR primer, without the need to proceeding to other devices again, avoids crossed contamination, and can complete quantitative analysis.
(3) the present invention is without the need to specific probe, without the need to order-checking, easy to use, and does not consume PCR primer in checkout procedure, can carry out downstream analysis further.
(4) high specificity of the present invention, can be good at distinguishing S. pullonum from Salmonella enteritidis and avian infectious bronchitis nephritis virus and other bacteriums, solves traditional detection method and differentiates the problem that S. pullonum accuracy rate is low.The present invention is highly sensitive, and lowest detection lower limit can reach 34 every microlitres of copy number (μ L).Wide application prospect is illustrated in the quick diagnosis of S. pullonum.
Accompanying drawing explanation
Fig. 1 is S. pullonum, Salmonella enteritidis and avian infectious bronchitis nephritis virus standard model HRM stdn melting curve figure.
Fig. 2 is S. pullonum, Salmonella enteritidis and avian infectious bronchitis nephritis virus and other bacterium standard model HRM peaks type melting curve figure.
Fig. 3 is specific detection and S. pullonum clinical sample detected peaks type melting curve figure.
Fig. 4 is S. pullonum sensitivity technique peak type melting curve figure.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1 primer
The Salmonellas whole genome sequence announced according to NCBI and consult related data and find S. pullonum (SalmonellaPullorum) 237 regular changes on rfbS gene, all white dysenterys are all G, and the serotype such as fowl typhoid, enteritis is A, the bacterial strain of other some serotypes and other kind is not containing this gene or change greatly, admittedly can according to this site design specific amplification primer.
Adopt Oligo7 primer-design software, according to Salmonellas rfbS sequence (the GeneBank accession number of the rfbS sequence of S. pullonum: LK931482 that GeneBank issues; The GeneBank accession number of the rfbS sequence of avian infectious bronchitis nephritis virus: AF442573; The GeneBank accession number of Salmonella enteritidis rfbS sequence: CP007267) and the rfbS sequence (this rfbS sequence and GeneBank accession number: in LK931482 identical) that obtains of S. pullonum reference culture order-checking, design primer pair in both sides, rfbS gene 237 site.Upstream primer rfbS-F:5 '-AAAGCAATATTCTTATGCCTA-3 '; Downstream primer rfbS-R:5 '-CACAATTTATGAATACTGCATC-3 '.Expanding fragment length is 72bp.
The preparation of embodiment 2 S. pullonum standard substance and PCR-HRM analyze
(1) extraction of DNA of bacteria:
Recovery S. pullonum reference culture (CVCC535), choose single bacterium colony and shake bacterium 12h in 1mLLB substratum, carry out DNA of bacteria extraction with OmegaDNAkit test kit, operation carries out the Isolation and purification of sample DNA, DNA-20 DEG C of preservation after extraction to specifications.
(2) preparation of standard substance:
In order to verify the feasibility of primer of the present invention and method, build positive simultaneously, there is provided positive template for clinical sample afterwards detects, with the reference culture DNA extracted for template, utilize the upstream primer rfbS-F described in embodiment 1, downstream primer rfbS-R carries out regular-PCR amplification.
Response procedures is 95 DEG C of denaturation 3min; 95 DEG C of sex change 20s, 55 DEG C of annealing 20s, 72 DEG C extend 30s; Cycle number 40 times; 72 DEG C of ends extend 3min.Obtain S. pullonum standard substance pcr amplification product after reaction, pcr amplification product is diluted 100 times, namely obtain S. pullonum standard model, for follow-up study.
(3) PCR-HRM amplification
Be template with DNA, utilize the upstream primer rfbS-F described in embodiment 1, downstream primer rfbS-R and saturated fluorescence dyestuff to carry out PCR-HRM amplification.
PCR-HRM reaction system is 10 μ L:
| PremixEx-Taq | 5.0μL |
| Primer rfbS-F | 0.5μL |
| Primer rfbS-R | 0.5μL |
| DNA profiling | 1.0μL |
| Eva green dyestuff | 1.0μL |
| ddH 2O | 2.0μL |
| Total | 10μL |
PCR-HRM response procedures is:
With the melting temperature (Tm) Tm value after the amplification of S. pullonum standard positive template for reference, testing sample melting temperature (Tm) Tm value the value of the confidence (GCP) is greater than 95% for S. pullonum.The melting temperature (Tm) of S. pullonum is: 70.32 ± 0.1 DEG C, fowl typhoid or Salmonella enteritidis: 69.5 ± 0.1 DEG C, and the amplification of other bacterial strains (other described bacterial strains are the bacterial strain of the sequence number 4 ~ 15 described in table 1) nothing, as Fig. 1,2.Wherein, the bacterial strain in table 1 all carries out DNA of bacteria extraction with OmegaDNAkit test kit.
The application of embodiment 3 S. pullonum PCR-HRM detection technique
From the ill poultry of doubtful bacteriological infection clinically, gather the diseased region samples such as liver spleen, ovary, uterine tube, collect 58 increment product altogether.
(1) process of clinical sample
From collecting the internal organs such as liver, spleen, ovary, uterine tube getting the tested chicken of 25g clinically, after grinding, sample joins aseptic increasing bacterium bag, add 225gBPW (protein water) 100r/min in 37 DEG C of constant-temperature tables and increase bacterium 6 ~ 8h in advance, get the pre-enrichment liquid of 1mL, add 9mL selective enrichment broth TTB (four sulphonic acid sodium are brilliant green), 37 DEG C of constant temperature culture 20 hours.
(2) extraction of sample DNA
Get the pre-enrichment liquid 1mL of selectivity, according to the operation of OmegaDNAkit test kit specification sheets, carry out the Isolation and purification of sample DNA, DNA-20 DEG C of preservation after extraction.
(3) PCR-HRM amplification
Be template with DNA, utilize the upstream primer rfbS-F described in embodiment 1, downstream primer rfbS-R and saturated fluorescence dyestuff to carry out PCR-HRM amplification.
PCR-HRM reaction system is 10 μ L:
| PremixEx-Taq | 5.0μL |
| Primer rfbS-F | 0.5μL |
| Primer rfbS-R | 0.5μL |
| DNA profiling | 1.0μL |
| Eva green dyestuff | 1.0μL |
| ddH 2O | 2.0μL |
| Total | 10μL |
PCR-HRM response procedures is:
With the melting temperature (Tm) Tm value after the amplification of S. pullonum standard positive template for reference, testing sample melting temperature (Tm) Tm value the value of the confidence (GCP) is greater than 95% for S. pullonum.With S. pullonum standard substance template for reference, all positive strain Tm values are all within 95% value the value of the confidence (GCP), and melting temperature (Tm) is within 70.32 ± 0.1 DEG C.
Then utilize traditional method to verify, the result goodness of fit is 100%.Namely in 58 increment product 29 parts be S. pullonum infect, 10 parts be avian infectious bronchitis nephritis virus or Salmonella enteritidis infection, 19 parts is other Salmonellass or other strain infections.As Fig. 3.
Embodiment 4 S. pullonum specific PCR-HRM detection method
Choose bacterium common in chicken body and the common serological type strain bacterial strain in contrast of Salmonellas, the PCR-HRM method according to setting up detects.Prove primer and the specificity of method of design, concrete bacterial strain and detected result as shown in table 1:
The detected result of table 115 kind of concrete bacterial strain
| Sequence number | Reference strain | Numbering | Result |
| 1 | Salmonella Pullorum (S. pullonum) | CVCC535 | + |
| 2 | Salmonella Gallinarum (avian infectious bronchitis nephritis virus) | CICC21510 | － |
| 3 | Salmonella Enteritidis (Salmonella enteritidis) | ATCC13076 | － |
| 4 | Salmonella Typhimurium (Salmonella typhimurium) | ATCC14028 | － |
| 5 | Salmonella Agona (Argonne receive Salmonellas) | ATCC51957 | － |
| 6 | Salmonella Weltevreden (Wei Taifuleideng Salmonellas) | ATCC BAA-2568 | － |
| 7 | Salmonella Meleagridis (turkey Salmonellas) | CICC21511 | － |
| 8 | Salmonella Thompson (Thompson Salmonellas) | ATCC8391 | － |
| 9 | Salmonella Senftenberg (mountain Fu Dengbao Salmonellas) | ATCC43845 | － |
| 10 | Salmonella Derby (Derby Salmonellas) | ATCC6960 | － |
| 11 | Salmonella Stanle (Stanley Salmonellas) | ATCC7308 | － |
| 12 | Salmonella Infantis (salmonella infantis) | ATCC BAA-1675 | － |
| 13 | Escherichia coli (intestinal bacteria) | ATCC25922 | － |
| 14 | Staphylococcus aureus (streptococcus aureus) | ATCC25923 | － |
| 15 | Pasteurella multocida (pasteurella multocida) | ATCC12948 | － |
The sensitivity technique of embodiment 5 S. pullonum PCR-HRM detection method
With regular-PCR method amplification reference culture S. pullonum rfbS gene (this rfbS sequence and GeneBank accession number: in LK931482 identical).PCR primer carries out 1.2% agarose gel electrophoresis analysis, adopt commercialization PCR primer glue recovery test kit (Omega) to instruct to specifications to reclaim PCR primer, PCR primer after purifying is connected on pMD19T carrier, product conversion will be connected to competent cell, competent cell after transforming is applied on ammonia benzyl resistance LB nutrient agar medium plate, after cultivating, picking list bacterium colony is cultivated in the LB nutrient solution containing ammonia benzyl resistance, extract plasmid after 16 ~ 18 hours and check order, confirming successful connection.Measure the concentration of the positive plasmid of successful connection, and dilute according to 10 times of coubling dilutions, obtain the standard model of different concns.
Be starting point concentration according to plasmid concentration after successful connection, the bottom line detected is that stoste dilutes 9 times, according to formula (6.02 × 10 (23) secondary copy numbers/mole) × (concentration)/(MWg/mol)=copies/mL carry out calculating to lowest detection lower limit be 34copies/ μ L, as Fig. 4.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (7)
1. a PCR-HRM primer for rapid detection S. pullonum, is characterized in that: its nucleotide sequence is as follows:
The complementary sequence of upstream primer rfbS-F:5 '-AAAGCAATATTCTTATGCCTA-3 ' or its Nucleotide;
Downstream primer rfbS-R:5 '-CACAATTTATGAATACTGCATC-3 ' or its nucleotide complementary sequence.
2. a PCR-HRM method for non-diagnostic object rapid detection S. pullonum, is characterized in that comprising the following steps:
A: extract DNA of bacteria;
B: be template with DNA, utilizes the upstream primer rfbS-F described in claim 1, downstream primer rfbS-R and saturated fluorescence dyestuff to carry out amplified reaction, obtains amplified production;
C: carry out HRM analysis to amplified production, determines whether as S. pullonum.
3. PCR-HRM method according to claim 2, is characterized in that:
The system of the pcr amplification reaction described in step B is:
4. the PCR-HRM method according to Claims 2 or 3, is characterized in that:
Described saturated fluorescence dyestuff is Evagreen dyestuff.
5. PCR-HRM method according to claim 2, is characterized in that:
The program of the amplified reaction described in step B is:
95 DEG C of denaturation 3min; 95 DEG C of sex change 20s, 53 ~ 56 DEG C of annealing 20s, 72 DEG C extend 20s; Circulate 40 times; 72 DEG C of ends extend 3min; 60 DEG C to 80 DEG C are carried out solubility curve analysis with the speed of 0.3 DEG C/step.
6. PCR-HRM method according to claim 2, is characterized in that:
The concrete analysis process that HRM described in step C analyzes is: with the melting temperature (Tm) Tm value after the amplification of S. pullonum standard positive template for reference, and testing sample melting temperature (Tm) Tm value the value of the confidence is greater than 95% for S. pullonum.
7. the PCR-HRM primer of rapid detection S. pullonum according to claim 1 is in the detection of S. pullonum, the purification of plant's S. pullonum, in Molecule Epidemiology Investigation or poultry farming S. pullonum risk assessment in application.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923343A (en) * | 2019-12-13 | 2020-03-27 | 华南农业大学 | Isothermal amplification primer group and kit for rapidly detecting salmonella pullorum |
| CN114395641A (en) * | 2022-01-19 | 2022-04-26 | 山东省农业科学院 | Primer pair and kit for detecting blast disease-resistant gene PigmR, application of primer pair and kit and detection method |
| CN117987579A (en) * | 2024-04-07 | 2024-05-07 | 华南农业大学 | Primer and probe for detecting salmonella pullorum, detection system and application |
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2015
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923343A (en) * | 2019-12-13 | 2020-03-27 | 华南农业大学 | Isothermal amplification primer group and kit for rapidly detecting salmonella pullorum |
| CN114395641A (en) * | 2022-01-19 | 2022-04-26 | 山东省农业科学院 | Primer pair and kit for detecting blast disease-resistant gene PigmR, application of primer pair and kit and detection method |
| CN117987579A (en) * | 2024-04-07 | 2024-05-07 | 华南农业大学 | Primer and probe for detecting salmonella pullorum, detection system and application |
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Application publication date: 20160203 |