CN101082061B - Highly pathogenic salmonella PCR rapid detecting probe - Google Patents
Highly pathogenic salmonella PCR rapid detecting probe Download PDFInfo
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- CN101082061B CN101082061B CN200610035803A CN200610035803A CN101082061B CN 101082061 B CN101082061 B CN 101082061B CN 200610035803 A CN200610035803 A CN 200610035803A CN 200610035803 A CN200610035803 A CN 200610035803A CN 101082061 B CN101082061 B CN 101082061B
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- pathogenic salmonella
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention discloses one kind of fast PCR detection probe for high pathogenic salmonella. The fast PCR detection probe includes the following gene seqs: P35 of F-5'-GGA AAT CGG ACC TAC GGG-3' and P36 of R-3'-GAC AGC CCG AGA CCC TAA-5'. The probe of the present invention can detect pathogenic salmonella containing toxic plasmid in the sample, and has high detection speed, high efficiency, high specificity and high sensitivity. The present invention is favorable to the early clinical diagnosis of salmonellosis of both human and animal, and the tracing and monitoring of pathogenic salmonella in food, water source and environment, and possesses important significance in practical detection of pathogenic salmonella in different fields.
Description
Technical field
The present invention relates to a kind of salmonella PCR and detect primer, be meant that especially the PCR that a kind of energy rapid detection has the highly pathogenic salmonella of toxicity plasmid detects primer.
Background technology
Salmonellas is to cause poisoning by food one of The main pathogenic fungi with food origin disease, and State Administration for Quality Supervision and Inspection and Quarantine clearly regulation Salmonellas is essential items for inspection.In the prior art, normally use all categories of PCR probe rapid detection salmonella or use the independent a kind of pathogenic salmonella of certain PCR probe in detecting.Its biggest advantage of these prior aries is: can utilize round pcr apace the kind of numerous salmonellas all to be detected, or apace a certain pathogenic salmonella be detected.But its shortcoming is: which the former can't distinguish in numerous Salmonellass be the kind that contains toxic plasmid, can cause disease, or even cause the important pathogen of infecting both domestic animals and human salmonellosis, and which is the kind with toxicity plasmid; The latter then can only detect a kind of pathogenic salmonella, detect the pathogenic salmonella that whether also contains other as need and then need to use instead corresponding PCR probe, common highly pathogenic salmonella has how many kinds of, then needs to change how many kinds of PCR probe, and detecting operation is inconvenience very.
Summary of the invention
Technical problem to be solved by this invention is: provide a kind of highly pathogenic salmonella PCR to detect primer, it can detect the various highly pathogenic salmonellas that contain in the sample efficiently, apace.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: provide a pair of highly pathogenic salmonella PCR to detect primer, this gene order to primer is as follows:
P35：5’-GGA?AAT?CGG?ACC?TAC?GGG-3’
P36：5’-AAT?CCC?AGA?GCC?CGA?CAG-3’。
The invention has the beneficial effects as follows: compared with prior art, primer of the present invention can detect having the toxicity plasmid, the Salmonellas of pathogenic effects is arranged in the sample fast and efficiently, specificity and susceptibility are good, help tracking monitor to pathogenic salmonella in the early clinical diagnosis of the common salmonellosis of humans and animals and food, water source, the environment, and for public health, food sanitation, herding once cured and port quarantine in detecting of pathogenic salmonella have important and practical meanings.
Description of drawings
Fig. 1 uses the detected result figure that highly pathogenic salmonella PCR of the present invention detects primer.
Embodiment
One, design of primers principle
Have data to show: the Salmonellas bacterial strain with toxicity plasmid to the virulence of mouse than its corresponding plasmid-free bacterial strain strong hundreds of extremely tens thousand of times do not wait, so far successively in mouse typhus, hog cholera, the Dublin, enteritis, the sheep miscarriage, fowl typhoid, identify the virulence plasmid of different molecular weight in the Salmonellass such as white dysentery, owing to there is the zone about the 10kb of a high conservative that contains the virulence gene in the virulence plasmid (spv) of different serotypes Salmonellas, therefore by detection, can identify the common strong pathogenic salmonella serotype that has to this conserved regions.
Sequence by the spvR gene in the toxicity plasmid that to understand modal Salmonellas with toxicity plasmid be which kind and these Salmonellass, and the sequence of the spvR gene of various toxicity plasmids compared, owing to have the zone about the 10kb of a high conservative that contains the virulence gene in the virulence plasmid (spv) of different serotypes Salmonellas, thereby design specific highly pathogenic salmonella PCR rapid detection primer of the present invention.
From NCBI (U.S. state-run biotechnology information center) search S.typhimurium (Salmonella typhimurium), S.choleraesuis (Salmonella choleraesuls), S.enteritidis (Salmonella enteritidis), the spvR gene order that each representative strain of S.galli-narum (white dysentery Salmonellas) is announced, and carry out Blast (NCBI development biological gene Database Systems) retrieval, and submit EBI (European molecular biology research institute) and utilize ClustalW (1.82) software to carry out the multiple sequence comparison.By Blast retrieval and multiple sequence comparison, find that the spvR gene order has very strong conservative property, consider in conjunction with multiplex PCR, utilize a pair of primer of Primer Premier 5 software designs, primer of the present invention is a spvR-P35-P36-PCR rapid detection primer, and this primer comprises following gene order:
P35：5’-GGA?AAT?CGG?ACC?TAC?GGG-3’
P36：5’-AAT?CCC?AGA?GCC?CGA?CAG-3’。
Two, confirmatory experiment
Utilize common Salmonellas reference cultures such as mouse typhus, hog cholera, enteritis, white dysentery, fowl typhoid, sheep miscarriage, verify the susceptibility of spvR-P35-P36-PCR rapid detection primer of the present invention with toxicity plasmid.
As shown in Figure 1, the bacterial strain kind of each code name representative is respectively among the figure: 1. Pparatyphoid A, 2. paratyphoid B, 3. mouse typhus, 6. hog cholera, 7. Newport, 9. Salmonella enteritidis, 12. white dysenterys, 20. fowl typhoids, 13. Salmonella anatis, 15. mountain Fu Dengbao, 16. A Baiding, 19. Jakarta, the miscarriage of 21. sheep, 22. colon bacillus, 24. proteus vulgaris, 26. serratia marcescens, 28. clostridium perfringens.Detected result shows that the Salmonellas reference culture of all spvR of containing has all obtained specific amplification, and amplified production is 507bp, and other Salmonellas reference culture that does not contain spvR then all shows negative.The result shows that spvR-P35-P36-PCR rapid detection primer of the present invention can be used as the PCR Auele Specific Primer that evaluation has toxicity plasmid Salmonellas fully.
Three, Application Example
With spvR-P35-P36-PCR rapid detection primer of the present invention, directly apply in the pcr amplification process of sample to be checked, its concrete enforcement follows these steps to carry out:
1.PCR reaction system (50 μ l)
Comprise following component:
Sterilized water: 14 μ l MgCl2:6 μ l dNTP:6 μ l
P35：4.5μl P36：4.5μl 10×buffer：5μl
Dna profiling solution: 7.5 μ l Taq enzymes: 2.5 μ l
Each component (not comprising dna profiling solution) is mixed the back divide and install to each PCR pipe, add mixing behind the dna profiling solution more respectively and obtain the PCR reaction system.
2.PCR reaction conditions
Tube＝0.5ml V＝50μl Lid＝105℃
1.T＝95℃×5min
2.T＝94℃×1min
3.T＝56℃×1min
4.T＝72℃×1.5min
5.Go?TO?2?For?32Cycle
6.T＝72℃×5min
7.Sound*
8.Hold?4℃?Forever
3.PCR product is identified
Prepare 2% plain agar sugar gel with 0.5 * tbe buffer liquid, press 0.5mg/L and add bromination second shallow lake (EB) glue.Get 10 μ l pcr amplification products, add point sample behind the 2 μ l Loading Dye mixings, 5 μ l DNAmarker.100V electrophoresis 20min is again in 80V electrophoresis 40min.
Utilize the Syngene gel imaging system to observe electrophoresis result, it is positive that specific amplification band person appears in the 507bp place, otherwise negative.
Claims (1)
1. a pair of highly pathogenic salmonella PCR detects primer, it is characterized in that this gene order to primer is as follows:
P35：5’-GGA?AAT?CGG?ACC?TAC?GGG-3’
P36：5’-AAT?CCC?AGA?GCC?CGA?CAG-3’。
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| CN200610035803A CN101082061B (en) | 2006-05-29 | 2006-05-29 | Highly pathogenic salmonella PCR rapid detecting probe |
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| CN200610035803A CN101082061B (en) | 2006-05-29 | 2006-05-29 | Highly pathogenic salmonella PCR rapid detecting probe |
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| CN101082061A CN101082061A (en) | 2007-12-05 |
| CN101082061B true CN101082061B (en) | 2010-05-12 |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101705307B (en) * | 2009-12-11 | 2012-07-18 | 上海交通大学 | PCR detection method of Salmonella typhimurium, nucleic acid and primer pair thereof |
| CN103243159A (en) * | 2013-04-11 | 2013-08-14 | 深圳职业技术学院 | Fluorescent PCR (polymerase chain reaction) detection probe, kit and detection method for Salmonella having virulence plasmids |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1743459A (en) * | 2004-08-20 | 2006-03-08 | 深圳太太基因工程有限公司 | Primer for detecting salmonella nucleotide fragment and probe sequence |
| CN1771331A (en) * | 2003-03-31 | 2006-05-10 | 科学和工业研究委员会 | stn gene oligonucleotide primers for detecting salmonella species and detection process using the same |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1771331A (en) * | 2003-03-31 | 2006-05-10 | 科学和工业研究委员会 | stn gene oligonucleotide primers for detecting salmonella species and detection process using the same |
| CN1743459A (en) * | 2004-08-20 | 2006-03-08 | 深圳太太基因工程有限公司 | Primer for detecting salmonella nucleotide fragment and probe sequence |
Non-Patent Citations (2)
| Title |
|---|
| 刘华伟等.畜禽及环境中沙门氏菌的PCR快速检测与控制.家畜生态学报26 （2）.2005,26(（2）),全文. |
| 刘华伟等.畜禽及环境中沙门氏菌的PCR快速检测与控制.家畜生态学报26 (2).2005,26((2)),全文. * |
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