CN1069925C - Salmonella rDNA spacer nucleic acid molecular probe and its using method - Google Patents
Salmonella rDNA spacer nucleic acid molecular probe and its using method Download PDFInfo
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Abstract
The present invention relates to an rDNA spacer ribonucleic acid molecular probe for detecting salmonella, which belongs to molecular biology. An rDNA central African RNA encoding region of salmonella is used as detection target genes, and five groups of 9 kinds of salmonella are used as development materials. The rDNA spacer ribonucleic acid molecular probe is synthesized by a conventional DNA synthesis method, the structure comprises 23 ribonucleotides, and the nucleotide sequence is 5'-CGAAGCATACATCAGTATGTTAG-3'. The probe can be used for detecting salmonella in various samples of foods, medicines, environment, etc. by a ribonucleic acid molecular hybridization method, a conventional PCR method and a reference primer PCR method, and furthermore, the probe has the advantages of simple and convenient operation, high speed, precision, sensitivity, no poison, etc.
Description
The present invention relates to molecular biology, specifically is exactly a kind of bioprobe that is used to check Salmonellas, and it can directly apply to inspection for food hygiene, the Salmonellas check in environment measuring, customs quarantine control and the clinical medicine.
The bacterial classification that belongs to Salmonellas is various, has found more than 2000 kinds it is common pathogenic bacterium so far.The Salmonellas check is the essential items for inspection of food sanitation and environment quarantine, at present, the Salmonellas method of inspection of China Ministry of Health promulgation, it mainly is form according to Salmonellas, physiological and biochemical property and sero-reaction are checked typing, its process complexity, operation are numerous tired, and the cycle is long, and the bacterium generation cross reaction that is close of Yi Yuqi and influence accuracy.In the existing Salmonellas method of inspection even also will use poisonous reagent (potassium hydride KH), staff's health there is harm.
Up to now, China does not have any research and patent about the Salmonellas nucleic acid molecular probe to deliver as yet, but developed country has all dropped into the development that substantial contribution and manpower are carried out the new method of inspection of Salmonellas and nucleic acid molecular probe in the world.In the rDNA nucleic acid molecular probe, comprise rRNA coding region probe and the different probe of non-RNA coding region probe two classes.A kind of Salmonellas rDNA probe is disclosed in United States Patent (USP) VS5147778, it is the target gene of detection with rRNA coding region sequence among rRNA or the rDNA, though it has set up the method for inspection of a kind of quick, nontoxic Salmonellas, sensitivity is still not high enough.And seek the higher detection of a target of specificity, and difficulty is very big, and the difficulty of development probe is just bigger.Researchist such as the U.S., Japan is developed to this direction.But at present, both at home and abroad all without any relevant research report.
The objective of the invention is to overcome the weak point that background technology exists,, develop higher nucleic acid molecular probe of a species specificity and application method thereof being the check that based gene detection and authenticate technology are applied to Salmonellas with the nucleic acid molecular probe.Set up a kind of quick, accurate, nontoxic highly sensitive Salmonellas check novel method that has again thus.
A kind of Salmonellas nucleic acid molecular probe, it is characterized in that making detection of a target gene in the coding region with non-RNA among the Salmonellas rDNA, design Salmonellas rDNA introns nucleic acid molecular probe, its structure is made up of 23 Nucleotide, and its nucleotide sequence is as follows: 5 ＇ CGAAGCATACATCAGTATGTTAG, 3 ＇.
RRNA and gene rDNA thereof are good target genes of development Salmonellas nucleic acid molecular probe, in rDNA, exist rRNA coding region and non-RNA coding region (being transcribed spacer), the rRNA coding region sequence can be used to develop Salmonellas rRNA probe and rDNA probe among rRNA sequence and the rDNA, and non-RNA coding region then can be used to develop Salmonellas rDNA introns nucleic acid molecular probe among the rDNA.
Bacterium rDNA transcribed spacer is a hypervariable region, and the rDNA spacer structure difference of different bacterium monoid is very big, and this difference can also reflect the distance (be that sibship is far away more, difference is big more, and vice versa) of sibship between the different bacterium.In the rDNA transcribed spacer, there are Salmonellas and the diverse nucleotide sequence of other non-Salmonellas, be Salmonellas feature nucleotide sequence, the Salmonellas rDNA introns nucleic acid molecular probe according to this characteristic sequence is set up has high Salmonellas specificity.The specificity reaction can take place with Salmonellas DNA in this probe, but with other non-Salmonellas DNA cross reaction does not take place, so can be used for evaluation of Salmonellas and the check of sample Salmonellas.
Set up Salmonellas rDNA introns nucleic acid molecular probe, at first to carry out molecular cloning and determined dna sequence to Salmonellas rDNA introns, and bacterial classifications different in the salmonella is investigated respectively, last result according to determined dna sequence and analysis, design and synthetic Salmonellas rDNA introns nucleic acid molecular probe, this probe can molecular hybridization or the mode of PCR testing sample is checked, as there is a Salmonellas, positive sign just appears, as not having Salmonellas, negative sign then appears.
The Salmonellas rDNA introns nucleotide sequence of having measured according to the contriver, and compare with other non-Salmonellas rDNA sequence data of having published.The present invention has selected five groups of 9 kinds of Salmonellass to be used to develop Salmonellas rDNA introns nucleic acid molecule probe.These five groups of 9 kinds of Salmonellass, bacterial classification all derives from quarantine station pathogenic bacterium chamber, Guangzhou, and their Chinese and Latin title are as follows:
First (A) group:
Salmonella paratyphi A (Salmonella paratyphi A)
Second (B) group:
Moscow' paratyphi B (S.paratyphi B)
Salmonella typhimurium (S.typhi murium)
Third (C1) group:
Moscow' paratyphi C (S.paratyphi C)
Moscow' tennessee (S.tennessee)
Fourth (D) group:
Salmonella typhi (S.typhi)
Salmonella enteritidis (S.enteritidis)
Penta (E1) group:
Salmonella anatis (S.anatum)
Wei Taifuleideng Salmonellas (S.weltevreden)
What the present invention set is made up of 23 Nucleotide, its nucleotides sequence is classified the Salmonellas rDNA introns nucleic acid molecular probe of 5 ＇ CGAAGCATACATCAGTATGTTAG 3 ' as, can adopt any conventional DNA synthetic method, produce in enormous quantities, for example: can use business-like automatic dna synthesizer synthetic.
The Salmonellas rDNA introns nucleic acid molecular probe of the present invention's development, can test for the Salmonellas in the various samples such as food, medical science, environment with making nucleic acid molecular hybridization method or conventional PCR method, the specificity reaction only takes place with Salmonellas DNA in this probe, and with non-Salmonellas DNA no cross reaction.It is distinctive with reference to the primer PCR method also to have set up a kind of the present invention in addition.
Introduce above-mentioned application method below respectively:
(1), making nucleic acid molecular hybridization method
Salmonellas rDNA introns nucleic acid molecular probe can be tested to the Salmonellas in the sample or a certain known bacterium is identified with the mode (for example dot blot mode) of various making nucleic acid molecular hybridizations.
The process of making nucleic acid molecular hybridization (comprising the pre-treatment of the mark and the testing sample of probe), molecular biology method routinely carries out.
Can be with nucleic acid hybridization to the sample direct survey in food, medical science, the environment, also can be earlier with the Salmonellas DNA cloning in the sample, checked again, have non-Salmonellas not disturb Salmonellas rDNA introns nucleic acid molecular probe that the specificity of Salmonellas is detected in the sample.
(2), conventional PCR method
Salmonellas rDNA introns nucleic acid molecular probe can be used as a kind of PCR primer and the pairing of another PCR primer, the PCR mode of bacterium rDNA universal primer for example, the Salmonellas DNA of trace in the check sample exists other non-Salmonellas DNA not disturb Salmonellas rDNA introns nucleic acid molecular probe that the Salmonellas specificity is detected in the sample quickly and accurately.
Can carry out according to conventional PCR working specification the process that Salmonellas carries out the PCR check with Salmonellas rDNA introns nucleic acid molecular probe.
(3), with reference to the primer PCR method
Adopt the different primer of a pair of above specificity in same test tube, same sample to be carried out PCR reaction, the target sequence difference that different primer amplifications amplifies, the molecular weight of its PCR product also has apparent difference by design.According to the kind of final PCR product in the test tube, can differentiate whether contain Salmonellas in the sample, or other bacterium and whether aseptic.Because the right PCR product reference each other of different primers is so be called with reference to the primer PCR method.PCR method result can the agarose gel electrophoresis check.
Be used for the Salmonellas check with reference to the primer PCR method, its PCR primer is selected to be provided with as follows:
1. do not hold conserved regions that a bacterium universal PC R primer is set with bacterium rDNA 16S rRNA coding region 3 ＇, it is that distance between Salmonellas rDNA introns nucleic acid molecular probe and the primer is 207 Nucleotide that the introns setting in rDNA has narrow spectrum another primer of Salmonellas.
2. do not hold conserved regions that a pair of bacterium class rDNA universal primer is set in addition at bacterium rDNA 16S rRNA coding region 5 ＇, the distance between them is 950 Nucleotide.
Fig. 1 electrophoresis result that five kinds of Salmonellass such as Salmonellas are hindered, trembled with fear to conventional PCR method with Salmonellas rDNA introns nucleic acid molecular probe check mouse for embodiment 1 promptly adopts.
Fig. 2 promptly adopts with reference to the electrophoresis result of primer PCR method with five kinds of Salmonellass such as Pparatyphoid A Sha Shi door bacterium of Salmonellas rDNA introns nucleic acid molecular probe check for example example 2.
The invention effect:
The invention of salmonella rDNA introns nucleic acid molecular probe and application process thereof has represented a kind of to sand The new method that the door Salmonella carries out identified for genes and detection, the method and existing conventional method are based on diverse Principle. New method has the easy advantage such as quick, accurate, sensitive, nontoxic. Can be used for strengthening or getting The conventional salmonella method of inspection for the promulgation of China Ministry of Public Health.
Compare with the existing salmonella method of inspection, the new method advantage is as follows:
1, easy to be quick: the routine inspection operation is numerous tired, experimental period long (sometimes will reach a week), New method only needs several hours (carry out the amplification of bacterium such as needs, proliferation time except).
2, accurate: salmonella rDNA introns nucleic acid molecular probe only with salmonella rDNA hybridization reaction, And with non-salmonella DNA cross reaction does not take place.
3, highly sensitive: salmonella rDNA introns nucleic acid molecular probe can be united with PCR method to be made With, theory value of detecting of salmonella can reach a bacterium.
4, do not use toxic reagent.
1, the preparation of nucleic acid molecular probe and PCR primer
1. classify 5 ' CGAAGCATACATCAGTATGTTAG 3 as by nucleotides sequence, select automatic dna synthesizer for use, adopt conventional DNA synthesis method, synthetic Salmonellas rDNA introns nucleic acid molecular probe.
2. Salmonellas rDNA introns nucleic acid molecular probe (Salmonellas specificity) and other three kinds of bacterium rDNA PCR universal primer P
1486(20 Nucleotide are long), P
16+(22 Nucleotide are long), P
16-(22 Nucleotide are long), all synthetic with automatic dna synthesizer, be dissolved in TE (Tris-Hcl Tomm, EDTA 1mmPH7.6) solution, concentration is 0.5 microgram/microlitre.
2, Salmonellas rDNA introns nucleic acid molecular probe check Salmonellas operating process (conventional PCR method)
1. Salmonellas sample pretreatment:
Get 1 milliliter and contain Salmonellas (10
5Bacterium) nutrient solution is put into 1.5 milliliters of little vials, centrifugal (5000 rev/mins) 2 minutes, the supernatant that inclines adds 0.2M sodium hydroxide an amount of (with dissolving bacterium dynamics) in tubule, add 500 microlitre TE solution (PH7.0) immediately and be diluted to 1000 bacterium/microlitre according to the bacterium number again.
2. composition in the 100 microlitre I PCR mixed solutions:
Salmonellas rDNA introns nucleic acid molecular probe 0.5 microgram
P
1486Nucleic acid molecular probe 0.5 microgram
10 times of Tap dna polymerase buffers agent, 10 microlitres
Tap archaeal dna polymerase 2.5 units
With the sterilized water constant volume is 100 microlitres
3. composition in the 100 microlitre II PCR mixed solutions
With bacterium rDNA PCR universal primer P
16S+, P
16S-Replace Salmonellas rDNA introns nucleic acid molecular probe and P respectively
1486, other composition is with I PCR mixed solution.
4. PCR operation
Get 9 0.5 milliliter of little vials, add I PCR mixed solution 20 microlitres in 1～8 pipe respectively, add mouse then respectively and hinder 5 kinds of Salmonellas samples such as Salmonellas and intestinal bacteria sample (contrast).In the 9th pipe, add II PCR mixed solution 20 microlitres, add intestinal bacteria sample (contrast) then.In each pipe, add 50 microlitre mineral oil, be put on the PCR instrument, carry out the PCR reaction by follow procedure:
94 ℃ 5 minutes, 94 ℃, 50 ℃ and 72 ℃ each 1 minute, and circulate continuously 30 times, then 72 ℃ 5 minutes.
After having reacted, every pipe adds 5 microlitre load sample liquid (30%Ficol 0.25% bromjophenol blue).
Get 4 microlitre point samples in 2% sepharose, electrophoresis result as shown in Figure 1.
Fig. 1 shows:
1,2,12:1Kb molecular weight gradient
3, Salmonella typhimurium (being equivalent to 40 bacterium amounts)
4, moscow' tennessee (being equivalent to 40 bacterium amounts)
5, sterilized water
6, Salmonella enteritidis (being equivalent to 40 bacterium amounts)
7, Salmonella anatis (being equivalent to 40 bacterium amounts)
8, Wei Taifuleideng Salmonellas (being equivalent to 40 bacterium amounts)
9, intestinal bacteria (being equivalent to 400 bacterium amounts)
10, intestinal bacteria (being equivalent to 40 bacterium amounts)
11, intestinal bacteria (being equivalent to 40 bacterium amounts)
Adopt Salmonellas rDNA introns nucleic acid molecular probe and P
1486It is right to form the PCR primer, five kinds of different Salmonellass such as mouse typhus (3,4,6,7, No. 8) all can accurately detect (positive signs that 207 Nucleotide occur) and with intestinal bacteria (9, No. 10) no cross reaction.Adopt P
16S+And P
16S-The PCR primer of forming is right, and the e. coli dna sample (No. 11) identical with No. 10 samples tested, and in contrast, the positive sign of 950 Nucleotide occurs, and the DNA of bacteria existence be described.
1, the preparation of nucleic acid molecular probe and PCR primer is with embodiment 1
2, with Salmonellas rDNA introns nucleic acid molecular probe check Salmonellas operation (with reference to the primer PCR method)
1. the Salmonellas sample pretreatment is with embodiment 1
2. 100 microlitre III PCR mixed solution compositions:
Salmonellas rDNA introns nucleic acid molecular probe 0.5 microgram
P
1486Nucleic acid molecular probe 0.5 microgram
P
16+Nucleic acid molecular probe 0.5 microgram
P
16-Nucleic acid molecular probe 0.5 microgram
10 times of Tap dna polymerase buffers agent, 10 microlitres
Tap archaeal dna polymerase 2.5 units
With the water constant volume is 100 microlitres
3. PCR operation
Get 9 0.5 milliliter of little vials, each adds the above-mentioned PCR mixed solution of 20 microlitres, adds various Salmonellas samples and intestinal bacteria sample or sterilized waters such as Salmonella paratyphi A then respectively, and every pipe is with 50 microlitre Dormant oils cappings, and schedule of operation is with embodiment 1.Electrophoresis result as shown in Figure 2.
Fig. 2 shows:
1, salmonella typhi (being equivalent to 40 bacterium amounts)
2, moscow' paratyphi C (being equivalent to 40 bacterium amounts)
3, salmonella typhi (being equivalent to 100 bacterium amounts)
4, moscow' paratyphi C (being equivalent to 100 bacterium amounts)
5, moscow' paratyphi B (being equivalent to 100 bacterium amounts)
6, Salmonella paratyphi A (being equivalent to 100 bacterium amounts)
7, Salmonella typhimurium (being equivalent to 40 bacterium amounts)
8, intestinal bacteria (being equivalent to 40 bacterium amounts)
11,1Kb molecular weight gradient
Adopting two pairs of primers is Salmonellas rDNA introns nucleic acid molecular probe/P
1486And P
16+/ P
16-With reference to the primer PCR method, the positive sign of Salmonellas of 207 Nucleotide can appear in sample at least that 1. contain Salmonellas, in addition, the positive for bacteria sign of another 950 Nucleotide can also occur, but in the sample that has, the positive for bacteria sign of 950 Nucleotide a little less than.2. do not contain Salmonellas, but containing the too sample of enterobacteria (No. 8), the positive for bacteria sign of 950 Nucleotide only occurring.3. in sterile sampling, there is not any DNA band (No. 9).
Claims (2)
1, Salmonellas nucleic acid molecular probe, it is characterized in that making detection of a target gene in the coding region with non-RNA among the Salmonellas rDNA, design Salmonellas rDNA introns nucleic acid molecular probe, its structure is made up of 23 Nucleotide, and its nucleotide sequence is as follows: 5 ＇ CGAAGCATACATCAGTATGTTAG3 ＇.
2, the application method of Salmonellas rDNA introns nucleic acid molecular probe, it is characterized in that forming by 23 Nucleotide, its sequence is that the probe of 5 ＇ CGAAGCATACATCAGTATGTTAG3 ＇ is used for Salmonellas when check, employing is with reference to the primer PCR method, its application method is to select the different primer of a pair of above specificity in same test tube same sample to be carried out the PCR reaction, and its PCR primer is selected to be provided with as follows:
1. do not hold conserved regions that a bacterium universal PC R primer is set with bacterium rDNA 16S rRNA coding region 3 ＇, it is that the distance that Salmonellas rDNA gives between nucleic acid molecular probe and the primer at interval is 207 Nucleotide that the introns setting in rDNA has narrow spectrum another primer of Salmonellas.
2. do not hold conserved regions that a pair of bacterium class rDNA universal primer is set in addition at bacterium rDNA 16S rRNA coding region 5 ＇, the distance between them is 950 Nucleotide.
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| CN95102601A CN1069925C (en) | 1995-03-10 | 1995-03-10 | Salmonella rDNA spacer nucleic acid molecular probe and its using method |
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|---|---|---|---|
| CN95102601A CN1069925C (en) | 1995-03-10 | 1995-03-10 | Salmonella rDNA spacer nucleic acid molecular probe and its using method |
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| CN1131194A CN1131194A (en) | 1996-09-18 |
| CN1069925C true CN1069925C (en) | 2001-08-22 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE69625769T2 (en) * | 1995-10-20 | 2003-08-07 | Institut Francais Du Petrole, Rueil Malmaison | Distributor for the independent introduction and / or discharge of fluids |
| CN100395347C (en) * | 2004-08-20 | 2008-06-18 | 深圳太太基因工程有限公司 | Primer for detecting salmonella nucleotide fragment and probe sequence |
| CN100478674C (en) * | 2006-05-30 | 2009-04-15 | 广州华峰生物科技有限公司 | Salmonella gene rapid diagnosis reagent kit based on annular mediated isothermal amplification technology |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989005359A1 (en) * | 1987-12-01 | 1989-06-15 | Integrated Genetics, Inc. | Detection of salmonella |
| US5147778A (en) * | 1987-12-01 | 1992-09-15 | Amoco Corporation | Probes and methods for the detection of salmonella |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989005359A1 (en) * | 1987-12-01 | 1989-06-15 | Integrated Genetics, Inc. | Detection of salmonella |
| US5147778A (en) * | 1987-12-01 | 1992-09-15 | Amoco Corporation | Probes and methods for the detection of salmonella |
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