CN1955311B - Nucleotide sequential, universal testing kit and method for detecting swine streptococcus - Google Patents

Abstract

The invention relates to a detection of Streptococcus suis nucleotide sequence, as follows : 1)5'-TAA CGG CTC ACC AAG GCT TC-3'(SEQ ID NO:1),2)5'-TTG CCG AAG ATT CCC TAC TG-3'(SEQ ID NO:2),3)5'-ATA CAT AGC CGA CCT GAG AGG GTG-3'(SEQ ID NO:3). The invention further provides a detection of Streptococcus suis type 2 kit and testing methods, inspection and quarantine areas belong. The invention of the advantages are : 1) Brief : shorten detection time of traditional methods of bacterial isolates from 3-7 days to 1.5 hours, 2) sensitivity : Sensitivity up 10-4, the conversion is equivalent to 100 CFU of bacteria can be detected ; 3) specific : Streptococcus suis bacteria other than the results are negative and found no cross-reaction 4) Stability : repeatability test results show that the establishment of good stability 5) Safety : Closed reaction, PCR without reprocessing, safe operation.

Description

Detect nucleotide sequence, general detection kit and the method for swine streptococcus

Technical field

The present invention relates to detect the nucleotide sequence of swine streptococcus, general detection kit and method, especially refer to a kind of fluorescence PCR detecting method fast and reagent thereof, be applicable to Streptococcus suis outburst epidemic situation is monitored in real time, can be widely used in the import and export and the animal-derived food product safety detection of animal product, belong to inspection and quarantine field.

Background technology

Swine streptococcus is an animal pathogen, can cause the pig meningitis, septicemia, and sacroiliitis, endocarditis is the common disease of piggy and feeder pig.This sick popular nothing is obviously seasonal, but summer, autumn pilosity, the characteristics of moist sultry weather pilosity are arranged.Sometimes even can be region outbursts, M ﹠ M is all very high, causes serious loss for livestock industry and international trade.According to kantigen, swine streptococcus can be divided into 35 serotypes, and 1/2,1,2,7,9 and 14 is the main serotype that activates the thing disease.Wherein streptococcus suis 2-type is important infecting both domestic animals and human disease pathogen, can cause people's meningitis, endocarditis and septicemia, and normal especially sending out in the working crowd who often contacts with live pig or pork product, hazardness is bigger.

Traditional swine streptococcus detection method is the bacterium partition method, and this method needs 3 days time at least, and length consuming time is necessary to develop a kind of method for quick.

Method for quick commonly used at present mainly is a molecular biology method, comprises nucleic acid hybridization, iso-electric point electrophoresis, oligonucleotide fingerprint map analyzing, polyacrylamide gel electrophoresis, polymerase chain reaction, monoclonal antibody technique etc.

Detection methods such as nucleic acid hybridization, iso-electric point electrophoresis, oligonucleotide fingerprint map analyzing, polyacrylamide gel electrophoresis, monoclonal antibody technique are applicable to carries out more deep research to the bacterium characteristic, reasons such as, cost height various owing to total system complicated operation, step are not suitable for carrying out simultaneously the detection of sample in enormous quantities.

The RT-PCR method specificity of common polymerase chain reaction is good, and the detection sensitivity height not only can detect live virus nucleic acid, also can directly detect the nucleic acid fragment of deactivation, the sample wide material sources, and suitability is good, is better than immunological method and bacterium partition method.What but this method was measured all is the end product of PCR, rather than the copy number of initiate dna or RNA.Owing to do not have linear relationship between the amount of the end product of PCR and the starting template amount,, can't accomplish accurately quantitative so can not calculate the copy number of initiate dna or RNA according to final PCR product amount.

Advantages such as the fluorescent quantitative PCR technique that emerges in recent years (also claiming the TaqMan technology) is highly sensitive with it, speed fast, high specificity are used widely at the aspects such as qualitative and detection by quantitative of gene expression dose analysis, pathogenic agent, and become the quantitative main method of current viral nucleic acid, we have applied it to the detection range of bird flu, Avian pneumo-encephalitis virus, have obtained good technical effect.

The main points of TaqMan technology are to increase specific fluorescence double-tagging probe on the original a pair of Auele Specific Primer of regular-PCR basis.This probe joint position is positioned at the centre in PBR territory.5 ' end of probe and the different fluorescein of 3 ' end difference mark, fluorescein as 5 ' end mark, the fluorescence that it sends can receive by detected instrument, be called report fluorophor (representing) with R, the fluorescein of 3 ' end mark, in closely, can absorb the fluorescent signal that 5 ' end report fluorophor sends, be called cancellation fluorophor (representing) with Q.When PCR was reflected at annealing stage, primer and probe combined with target gene fragment simultaneously, and the fluorescent signal that the R group sends on this moment probe is absorbed by the Q group, and instrument detecting is less than fluorescent signal that R sent; The PCR reaction proceeds to extension during the stage, and the Taq enzyme is under the guiding of primer, along the synthetic new chain of template strand; When the extension of chain proceeds to the probe joint position, the function of its 5 ' → 3 ' exonuclease of Taq enzyme performance of this moment, probe is cut into mononucleotide, and the R group that meanwhile is marked on the probe dissociates out, and or else the fluorescence that R sent received by Q absorbs detected instrument.

PCR carries out a circulation, when having synthesized the new chain of N bar, with regard to hydrolysis N bar probe, also discharged the fluorophor of respective numbers.The amount of received fluorescence signal intensity of instrument and PCR reaction product is corresponding relation.Along with moving in circles of PCR reaction, the PCR product is exponential form and increases the also corresponding growth of fluorescent signal.If measured fluorescent value is an ordinate zou during with each PCR loop ends, be X-coordinate mapping with the PCR cycle number, can obtain a curve that connects each circulation back fluorescent value-be called amplification curve.When containing the nucleotide sequence that will detect pathogenic agent to some extent in detecting sample, resulting curve is " S " type; And in sample, do not contain pathogenic agent, and then the PCR process does not take place, and probe is not hydrolyzed, and does not produce fluorescent signal, and its amplification curve is a sea line.

The pcr amplification signal enters the lower limit of relatively stable increased logarithmic phase, is set in usually near the growth flex point place of S type amplification curve, is called threshold line (Threshold); And the cycle number in amplification curve and threshold line point of crossing is called the Ct value.The concentration of pathogenic agent is high more in the sample, and the Ct value is just more little.Measure pathogen nucleic acid in the key sample not with this method, can not only fast qualitative, also because advanced fluorescent signal detection system of fluorescent PCR itself and powerful information processing capability, can realize quantitative to pathogen nucleic acid.

The present invention is applied to the swine streptococcus detection range with the fluorescent PCR technology first, and specific primer sequence, probe sequence, test kit and detection method at swine streptococcus are provided.

Technology contents

First purpose of the present invention provides that a group-specific is strong, highly sensitive, the nucleotide sequence of detection swine streptococcus with versatility, comprises primer sequence and probe sequence.

Another object of the present invention provides the test kit of a kind of detection swine streptococcus quick, accurate, easy to use.

But the 3rd purpose of the present invention provides the method for a kind of quick, accurate detection by quantitative swine streptococcus.

For achieving the above object, the present invention is by the following technical solutions:

The present invention selects swine streptococcus 16S rDNA as target region, design primer and probe.Primer length is about 20 bases, and GC content is 50%-60%, no secondary structure and repeatability in the primer, and with the interior no complementary sequence of primer, the melting temperature (Tm) between primer (Tm value) differs less than 5 ℃ between primer.The length of probe is about 25 bases, and the Tm value is worth high about 5 ℃ than primer Tm.

One group of nucleotide sequence that detects swine streptococcus, as follows:

1)5’-TAA CGG CTC ACC AAG GCT TC-3’(SEQ ID NO：1)

2)5’-TTG CCG AAG ATT CCC TAC TG-3’(SEQ ID NO：2)

3)5’-[FAM]ATA CAT AGC CGA CCT GAG AGG GTG[TAMRA]-3’(SEQ ID NO：3)

Wherein sequence 1) and 2) be respectively sense primer and antisense primer, sequence 3) be fluorescent probe, 5 ' end mark report fluorophor FAM (6-Fluoresceincarboxylic acid) of probe, 3 ' end mark cancellation fluorophor TAMRA.

We adopt TaqMan fluorescent PCR detection technique to set up general swine streptococcus detection method, the various conditions of reaction are optimized, comprising the screening of primer probe, Mg ²⁺The optimization of working concentration, the optimization of primer concentration and probe concentration obtains following test kit.

A kind of common reagent box that detects swine streptococcus ,-20 ℃ of preservations, composed of the following components:

1) DNA extraction reagent: available from INVITROGEN company, article No. 10503-027 carries out packing by the 5ml/ pipe, 6 pipe/boxes.

2) PCR reaction solution, 750 μ L * 1 pipe, see the following form:

Table 1

Component	Final concentration
		10 * PCR damping fluid	1 * PCR damping fluid
25mM MgCL 2	3.0mM
		2.0mM dNTP	0.2mM dNTP
Primer
	1	0.3μmol/L
Primer 2			0.3μmol/L
	Probe	0.1μmol/L

10 * PCR damping fluid, 25mM MgCl ₂, 2.0mM dNTP all purchases the company in Promega; It is synthetic that primer and probe all entrust Shanghai to dodge brilliant molecular biotechnology company limited, primer 1 has the nucleotide sequence shown in the sequence table SEQ ID NO:1, primer 2 has the nucleotide sequence shown in the sequence table SEQ ID NO:2, probe has the nucleotide sequence shown in the sequence table SEQ ID NO:3,5 ' end mark report fluorophor FAM of probe, 3 ' end mark cancellation fluorophor TAMRA; Consisting of of 10 * PCR damping fluid: 500mM KCl, 100mMTris-HCl (25 ℃ of pH9.0), 1.0%Triton X-100.

3) Taq archaeal dna polymerase 5U/ μ L, 15 μ L * 1 pipe, available from Promega company, article No. M1661;

4) the sterilization purified water of no DNA enzyme, 1mL * 1 pipe with twice distillation of tap water, through Millipore MILLI-QPF PLUS pure water instrument purifying, collects the water of resistivity 〉=18.0M Ω .cm, stores in the aseptic bottle.Millipore-Q purified water 151bf/in ²(1.034 * 10 ⁵Pa) high pressure steam sterilization is 15 minutes.

5) negative control: 1mL * 1 pipe, the aseptic SPF pig substantial viscera of winning is made 20% suspension with 0.01mol/L pH7.2 PBS buffer saline.60 ℃ of effects deactivation in 1 hour.

6) positive control: 1mL * 1 pipe, for containing the recombinant plasmid of swine streptococcus 16S rDNA specific fragment, the Ct value is 22.0～28.0.

The preparation method of positive control is: select swine streptococcus 16S rDNA as target region, adopt the primer sequence shown in SEQ ID NO:1 and the SEQ ID NO:2 that swine streptococcus is carried out the PCR reaction, reclaim pcr amplification product, obtain the specific fragment (all swine streptococcus all have this conserved sequence) of 114bp, after reclaiming purifying, be connected with PGEM-T easy carrier (purchasing company) in Promega, transform JM109 competent cell (purchasing company) in Promega, alkaline lysis method of extracting plasmid DNA, cut through PCR and enzyme and to identify the positive recombinant plasmid of back acquisition, called after PGEM-16S rDNA.10 times of gradient dilution lysates are measured the Ct value and are 22.0～28.0 and are positive control.

114 bp purpose fragment gene sequences are as follows:

1 TAACGGCTCA CCAAGGCATC GATACATAGC CGACCTGAGA GGGTGATCGG CCACACTGGG

61 ACTGAGACAC GGCCCAGACT CCTACGGGAG GCAGCAGTAG GGAATCTTCG GCAA

Synthetic primer and probe are HPLC analyze, as the ratio that obtains single absorption peak collection of illustrative plates and measure OD260nm/OD280nm with ultraviolet spectrophotometer promptly is considered as qualified primer between 1.6-2.0.The DNA that extracts is that template increases from the pig 2 type suis cultures of dilution in 1: 100, and the result shows for the swine streptococcus universal test method, and primer provided by the invention pair and probe are used, and the Ct value of amplification is relatively low, and increasing degree is higher.

The primer concentration that screening is good is that spacing increases progressively from 0.1 μ M to 0.8 μ M with 0.1 μ M, and concentration and probe concentration increases progressively with 0.025 μ M from 0.025 μ M to 0.2 μ M.Proportioning to primer and probe different concns compares, from repeatedly finding the revision test: the primer of different concns, probe are for this positive template, the CT value is basicly stable about 20.0, but primer concentration is 0.3 μ M, fluorescence amplification was higher relatively when concentration and probe concentration was 0.1 μ M, so selected primer concentration is 0.3 μ M, concentration and probe concentration is primer and the concentration and probe concentration of 0.1 μ M as swine streptococcus universal PC R detection method.

We are at Roche Light Cycler fluorescent PCR detector, after the pre-sex change of 92 ℃/3min, in this experiment denaturation temperature and time, annealing and elongating temperature and time having been carried out optimization experiment.During amplification, adopt cycle index to be respectively 40,45 and 50, the relatively Ct value of amplification, and then the cycle index of definite amplification.Final definite employing the shortest scheme consuming time is the PCR reaction parameter: 92 ℃/3min; 92 ℃/5sec, 60 ℃/30sec, fluorescence is collected in 45 circulations during each loop ends.

Studies show that Mg ²⁺Concentration is bigger to the influence of fluorescent PCR amplification, thus use the good primer probe of screening, with Mg ²⁺Concentration be that spacing increases progressively with 0.5mM from 1.5mM to 6.0mM, to different Mg ²⁺Amplification under the concentration conditions compares, and the result shows Mg ²⁺Concentration is relevant to the sensitivity of fluorescence increment and detection, improves its working concentration, can improve the fluorescence increment of detection probes, but along with Mg ²⁺The raising of concentration when particularly surpassing 5mM, when detecting some sample, can be found the phenomenon of floating on the curve.For universal test method, the Mg after the optimization ²⁺Concentration is 3.0mM.

The Taq enzyme that we select Promega company to produce, the definition of a unit: 74 ℃ act on 30 minutes, the dNTPs of 10nM can be mixed needed enzyme amount in the acid soluble material.The active requirement: tool dna polymerase activity and 5 ' → 3 ' exonuclease activity, do not have 3 ' → 5 ' exonuclease activity and endonuclease activity; The tool thermostability, 94 ℃ still kept 50% activity after 1 hour.The optimization experiment template of Taq enzyme dosage (in the U of unit) adopts 2 type suis cultures (dilution in 1: 100) to extract DNA and increases.From repeatedly selecting 1.0U Taq enzyme as the Taq enzyme amount of using the revision test result.

1) sample preparation: for the live pig sample, the brush,throat sampling will be goed deep into swab larynx and palate and scrape back and forth 3 times～5 times during sampling, get the throat juice.For tonsilla, internal organ or muscle samples, fully grind in mortar with aseptic scissors and tweezers clip sample 1.0g to be checked, add 5mL PBS mixing again, then tissue suspension is changed in the aseptic Eppendorf pipe, number standby.For serum or blood plasma, directly draw to aseptic Eppendorf pipe with asepsis injector, number standby.The sample of gathering or handling is preserved under 2 ℃～8 ℃ conditions and should be no more than 24h; If need prolonged preservation, must place-70 ℃ of refrigerators, but should avoid multigelation (freeze thawing is 3 times at most).After the sample sealing of gathering, adopt thermo jug or insulated tank sealing on the rocks, be transported to the laboratory as early as possible.

2) extraction of DNA: carry out in the sample process district.

Get n 1.5mL sterilization Eppendorf pipe, wherein n is sample number to be checked, a pipe positive control and a pipe negative control sum, and each pipe is numbered mark.

Every pipe adds the 1.0mL lysate, adds each 100 μ L of sample to be tested, negative control and positive control then respectively, and a sample is used a suction nozzle instead; Concussion mixing 5s on the vortex mixer.Under 4 ℃～25 ℃ conditions, the centrifugal 10min of 10 000r/min.

Get the 1.5mL sterilization Eppendorf pipe of equal amts, add 500 μ L dehydrated alcohols (20 ℃ of precoolings), each pipe is numbered.The supernatant liquor of drawing in each pipe of centrifugal back is transferred in the corresponding pipe, and supernatant liquor will fully be drawn, and the sucking-off bottom settlings is not put upside down mixing.

Under 4 ℃～25 ℃ conditions, the centrifugal 5min of 5 000r/min (the Eppendorf tube opening keeps placing towards the centrifugal basket direction of principal axis).Remove supernatant gently, be inverted on the thieving paper, blot liquid, different samples should blot in the different places of thieving paper.Add 1.0mL 75% ethanol, put upside down washing.

Under 4 ℃～25 ℃ conditions, the centrifugal 10min of 5 000r/mi n (the Eppendorf tube opening keeps placing towards the centrifugal basket direction of principal axis).Remove supernatant liquor gently, be inverted on the thieving paper, blot liquid, different samples should blot in the different places of thieving paper.

The centrifugal 10s of 4 000r/min is thrown to the pipe bottom with the residual liquid on the tube wall, it is blotted with micro sample adding appliance as far as possible, and a sample is used a suction nozzle instead, and suction nozzle is not run into precipitation one side, drying at room temperature 5s～15s.Should not be too dry, in order to avoid DNA is insoluble.

Adding 11 μ L does not have the sterilization purified water of DNA enzyme, mixing gently, and the DNA on the dissolving tube wall, the centrifugal 5s of 2 000r/min preserves standby on ice.The DNA that extracts must carry out pcr amplification or be positioned over-70 ℃ of refrigerators in 2h, nucleic acid is transferred to the reaction mixture preparation area.

3) pcr amplification: amplifing reagent is prepared and preparation, carries out at the reaction mixture preparation area.From test kit, take out the general fluorescent PCR reaction solution of swine streptococcus, Taq enzyme, after at room temperature melting, the centrifugal 5s of 2 000r/min.If required PCR number is n, wherein n is sample number to be checked, a pipe positive control and a pipe negative control sum.Each test reaction system need be used 15 μ L PCR reaction solutions and 0.3 μ L Taq enzyme.Calculate the usage quantity of each reagent, add in the proper volume test tube, each packing 15 μ L is transferred to the sample process district in each PCR pipe.Application of sample carries out in the sample process district.In the PCR of each setting pipe, add the dna solution 10 μ L of preparation respectively, make cumulative volume reach 25 μ L.Behind the tight pipe lid of lid, the centrifugal 30s of 500r/min.Fluorescent PCR is reflected at detection zone to carry out.PCR pipe behind the application of sample is put into the fluorescent PCR detector, and the record sample is put order.

Reaction parameter is provided with:

--fs, pre-92 ℃/3min of sex change;

--subordinate phase, 92 ℃/5s, 60 ℃/30s, 45 circulations, phosphor collection is arranged on when the each round-robin annealing of subordinate phase is extended and carries out.

4) result's judgement: the interpretation of result condition enactment, read detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve.Different instruments can be adjusted according to instrument noise situation.Quality control standard, negative control do not have the Ct value and do not have amplification curve.The Ct value of positive control answers≤30.0, and specific amplification curve occurs.As negative control and the discontented condition that is enough to of positive condition, it is invalid that experiment this time is considered as.The result describes and judges, feminine gender, and no Ct value and do not have amplification curve, showing does not have swine streptococcus in the sample; The positive, Ct value≤30.0, and specific amplification curve appears, there is swine streptococcus in the expression sample.

A kind of method that detects swine streptococcus comprises the steps:

1) extracts sample DNA;

2) sample DNA that extracts is carried out pcr amplification:

Primer sequence is 5 '-TAA CGG CTC ACC AAG GCT TC-3 '

5’-TTG CCG AAG ATT CCC TAC TG-3’

Fluorescent probe is 5 '-[FAM] ATA CAT AGC CGA CCT GAG AGG GTG[TAMRA]-3 ';

Amplification condition is 92 ℃/3min; 92 ℃/5sec, 60 ℃/30sec, fluorescence is collected in 45 circulations during each loop ends.

3) whether the fluorescence intensity of mensuration PCR reaction system exists swine streptococcus in the judgement sample; Feminine gender, no Ct value and do not have amplification curve, showing does not have swine streptococcus in the sample; The positive, Ct value≤30.0, and specific amplification curve appears, there is swine streptococcus in the expression sample.

Advantage of the present invention is: the present invention selects suis 16S rDNA as target region, and design primer and probe are set up and optimized the general fluorescence PCR detecting method of swine streptococcus, have obtained excellent technique effect:

1) quick: this method is monitored in real time to the PCR product, and PCR finishes to obtain the result, and 3～7 day detection time of traditional bacterium separation method shortened to 1.5 hours.

2) sensitivity: owing to adopted specific fluorescent probe and highly sensitive fluorescent PCR instrument, make this method highly sensitive 100～1000 times than traditional PCR method; With the method for being set up 4 strain streptococcus suis 2-type cultures with 10 times of gradient dilutions are detected, display sensitivity can reach 10 as a result ^-4, the bacterium that is equivalent to 100 CFU through conversion can detect;

3) special: owing to not only adopted specific primer, and adopted specific probe, make the specificity of this method be higher than conventional P CR method; The streptococcus suis 2-type fluorescence PCR detecting method of setting up is negative in the result who detects collected swine streptococcus and other bacterium, does not find cross reaction;

4) stable: the replica test result shows having good stability of institute's establishment method;

5) safety: totally-enclosed reaction need not the PCR aftertreatment, operational safety.

The invention will be further described below in conjunction with specification drawings and specific embodiments.

Description of drawings

Fig. 1 is the limit of detection measurement result of swine streptococcus universal test method.

Fig. 2 is that universal test method is to the streptococcic detected result of four strains.

Fig. 3 is the detected result of universal test method to other bacterium.

Fig. 4 is the detected result of brush,throat with universal detection method.

Fig. 5 is the detected result of universal detection method to pig tonsil.

Embodiment

The preparation of embodiment 1, test kit and use

1, the preparation of test kit is formed, and sees Table 2.

The preparation of table 2 test kit is formed

Form by (48tests/ box)	Quantity
		DNA extraction liquid	5.0mL * 6 pipes
The general fluorescent PCR reaction solution of swine streptococcus	750 μ L * 1 pipe
		Taq enzyme (5U/ μ L)	15 μ L * 1 pipe
The sterilization purified water of no DNA enzyme	1mL * 1 pipe
		Negative control	1mL * 1 pipe
Positive control	1mL * 1 pipe

2, using method

1) DNA extraction

Get n 1.5mL sterilization Eppendorf pipe, wherein n is sample number to be checked, a pipe positive control and a pipe negative control sum, and each pipe is numbered mark.

Every pipe adds the 1.0mL lysate, adds each 100 μ L of sample to be tested, negative control and positive control then respectively, and a sample is used a suction nozzle instead; Concussion mixing 5s on the vortex mixer.Under 4 ℃～25 ℃ conditions, the centrifugal 10min of 10 000r/min.

Get the 1.5mL sterilization Eppendorf pipe of equal amts, add 500 μ L dehydrated alcohols (20 ℃ of precoolings), each pipe is numbered.The supernatant liquor of drawing in each pipe of centrifugal back is transferred in the corresponding pipe, and supernatant liquor will fully be drawn, and the sucking-off bottom settlings is not put upside down mixing.

Under 4 ℃～25 ℃ conditions, the centrifugal 5min of 5 000r/min (the Eppendorf tube opening keeps placing towards the centrifugal basket direction of principal axis).Remove supernatant gently, be inverted on the thieving paper, blot liquid, different samples should blot in the different places of thieving paper.Add 1.0mL 75% ethanol, put upside down washing.

Under 4 ℃～25 ℃ conditions, the centrifugal 10min of 5 000r/min (the Eppendorf tube opening keeps placing towards the centrifugal basket direction of principal axis).Remove supernatant liquor gently, be inverted on the thieving paper, blot liquid, different samples should blot in the different places of thieving paper.

The centrifugal 10s of 4 000r/min is thrown to the pipe bottom with the residual liquid on the tube wall, it is blotted with micro sample adding appliance as far as possible, and a sample is used a suction nozzle instead, and suction nozzle is not run into precipitation one side, drying at room temperature 5s～15s.Should not be too dry, in order to avoid DNA is insoluble.

Adding 11 μ L does not have the sterilization purified water of DNA enzyme, mixing gently, and the DNA on the dissolving tube wall, the centrifugal 5s of 2 000r/min preserves standby on ice.The DNA that extracts must carry out pcr amplification or be positioned over-70 ℃ of refrigerators in 2h, nucleic acid is transferred to the reaction mixture preparation area.

2) amplifing reagent is prepared and preparation

Carry out at the reaction mixture preparation area.From test kit, take out the general fluorescent PCR reaction solution of swine streptococcus, Taq enzyme, after at room temperature melting, the centrifugal 5s of 2 000r/min.If required PCR number is n, wherein n is sample number to be checked, a pipe positive control and a pipe negative control sum.Each test reaction system need be used 15 μ L PCR reaction solutions and 0.3 μ LTaq enzyme.Calculate the usage quantity of each reagent, add in the proper volume test tube, each packing 15 μ L is transferred to the sample process district in each PCR pipe.Application of sample carries out in the sample process district.In the PCR of each setting pipe, add the dna solution 10 μ L of preparation respectively, make cumulative volume reach 25 μ L.Behind the tight pipe lid of lid, the centrifugal 30s of 500r/min.Fluorescent PCR is reflected at detection zone to carry out.PCR pipe behind the application of sample is put into the fluorescent PCR detector, and the record sample is put order.

Reaction parameter is provided with:

--fs, pre-92 ℃/3min of sex change;

--subordinate phase, 92 ℃/5s, 60 ℃/30s, 45 circulations, phosphor collection is arranged on when the each round-robin annealing of subordinate phase is extended and carries out.

3) result judges

The interpretation of result condition enactment reads detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve.Different instruments can be adjusted according to instrument noise situation.Quality control standard, negative control do not have the Ct value and do not have amplification curve.The Ct value of positive control answers≤30.0, and specific amplification curve occurs.As negative control and the discontented condition that is enough to of positive condition, it is invalid that experiment this time is considered as.The result describes and judges, feminine gender, and no Ct value and do not have amplification curve, showing does not have swine streptococcus in the sample; The positive, Ct value≤30.0, and specific amplification curve appears, there is swine streptococcus in the expression sample.

The sensitivity test of embodiment 2, test kit

1, material:

The bacterial isolates and the source that are applied in the table 3 method research process

Bacterial classification	Bacterial strain	The source or the unit of providing
			Streptococcus suis 2-type streptococcus suis 2-type streptococcus suis 2-type D group swine streptococcus	22 strains of Sichuan strain isolated 1 Sichuan strain isolated, C 55606And C 55953Lan Shi D group, C 55914	China Veterinery Drug Inspection Office provides, and is located away from the different areas, Sichuan and dies of illness in the pig body, with China Veterinery Drug Inspection Office of martin's bouillon recovery China Veterinery Drug Inspection Office

2, method

The martin's bouillon culture of the streptococcus suis 2-type of 4 strain heat inactivations and non-deactivation is done 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8, 10 ^-9, 10 ^-10Doubly the general fluorescent PCR of swine streptococcus is done in dilution.When measuring limit of detection, relatively heat inactivation is to the influence of fluorescence PCR detecting method.

Measure the CFU/ml of 2 strain streptococcus suis 2-type Sichuan strain isolateds, be about to the not streptococcus suis 2-type of deactivation of each extent of dilution, get the 0.1ml inoculation and use martin's bouillon, place 37 ℃ of hatchings to observe in 20-24 hour, the high dilution of long bacterium, muddiness.The general fluorescent PCR detected result of detected result and swine streptococcus compares.

3, result

See shown in Figure 1, the martin's bouillon culture of the streptococcus suis 2-type of deactivation or non-deactivation is made 10 times of serial dilutions, fluorescent PCR condition after utilization is optimized detects, and test-results shows that the limit of detection of 4 strain deactivations or the universal detection method of non-deactivation swine streptococcus can reach 10 ^-5, and this measurement result of testing with suis CFU is 10 ⁷/ ml shows that the limit of detection of fluorescence PCR method can reach 10～100 CFU.The result shows that also heat inactivation does not influence the sensitivity that fluorescent PCR detects.

The specificity test of embodiment 3, test kit

1, material

The bacterial isolates and the source that are applied in the table 4 method research process

Bacterial classification	Bacterial strain	The source or the unit of providing
			Streptococcus suis 2-type streptococcus suis 2-type streptococcus suis 2-type D group swine streptococcus streptococcus pneumoniae pasteurella multocida	22 strains of Sichuan strain isolated 1 Sichuan strain isolated, C 55606And C 55953Lan Shi D group, C 55914ATCC49619 1 strain	China Veterinery Drug Inspection Office provides, be located away from the different areas, Sichuan and die of illness in the pig body, with Beijing Administration for Entry-Exit Inspection and Quarantine of medicine biotechnology development company of Nat'l Pharmaceutical ﹠ Biological Products Control Institute of China Veterinery Drug Inspection Office of martin's bouillon recovery China Veterinery Drug Inspection Office moving inspection laboratory

The crooked bacterium of intestinal bacteria Salmonellas campylobacter jejuni colon

2 strains, 3 strains, 2 strains, 1 strain

Moving inspection laboratory China Veterinery Drug Inspection Office of China Veterinery Drug Inspection Office of China Veterinery Drug Inspection Office of China Veterinery Drug Inspection Office of Beijing Administration for Entry-Exit Inspection and Quarantine

2, method

1) detects different suis

With setting up the streptococcus suis 2-type of general fluorescence PCR detecting method to collecting, D group swine streptococcus and hyopneumoniae suis detect, and whether can detect other suis with verification method.

2) detect other various bacteriums

With the pasteurella multocida of general fluorescence PCR detecting method to collecting of setting up, intestinal bacteria, Salmonellas, campylobacter jejuni, the crooked bacterium of colon detects, with the specificity of verification method.

3, result

1) to other streptococcic detected results: as shown in Figure 2, it is all positive that universal test method of the present invention detects four collected strain suis.

2) with the pasteurella multocida of test kit of the present invention to collecting, intestinal bacteria, Salmonellas, campylobacter jejuni, the crooked bacterium of colon detects, and the result shows all negative (Fig. 3), shows that the method specificity of foundation is good.

Embodiment 4, the general fluorescence PCR method of swine streptococcus batch between, batch in repeatable test

For between this test kit is criticized, batch in repeatability comprehensively examine, we select three batches of qualified reagent carried out batch between, batch in repeatable test.

1, experiment material

Reagent: 3 batches of the general fluorescence PCR detection reagent kits of swine streptococcus, lot number: 200508001T, 200508002T, 200508003T

Detecting instrument: full-automatic fluorescent PCR detector ROCHE company product, model, LightCyclerII.

Detect sample: will be through heat-killed streptococcus suis 2-type bacterial strain C ₅₅₆₀₆The martin's bouillon culture do 10 ^-1, 10 ^-2, 10 ^-3Doubly after the dilution, between continuous criticize with the universal fluorescence PCR detecting method of swine streptococcus respectively for three times, criticize interior replica test.

2, experiment content: the every batch of reagent is all to through heat-killed streptococcus suis 2-type bacterial strain C ₅₅₆₀₆3 extent of dilution of martin's bouillon culture carry out 3 fluorescent PCRs and detect, 10 parts of multiple pipes of each pattern detection at every turn, then according to above data computation test kit batch in, batch between error.

Detected result requires: same sample batch in, batch between CV value≤10%

3, experimental result

With streptococcus suis 2-type bacterial strain C ₅₅₆₀₆Martin's bouillon culture heat inactivation, by 10 times of gradient dilutions is that 7 concentration (are labeled as J1, J2, J3, J4, J5, J6, J7 respectively, J0 is undiluted nutrient solution), each concentration is all used the universal fluorescence PCR detection reagent kit of swine streptococcus to carry out multiple pipe and is detected, and the result is as follows:

Table 5 streptococcus suis 2-type bacterial strain C ₅₅₆₀₆Martin's bouillon culture dilution result

The dilution gradient	J0	J1	J2	J3	J4	J5	J6	J7
										1	6.43	17.45	20.37	23.56	26.25	29.45	32.57	36.47
2	6.47	17.38	20.54	23.38	26.96	29.56	32.79	36.45

According to The above results, the martin's bouillon culture of selecting J1, J2, three concentration of J3 is as detecting sample.The martin's bouillon culture of J1, J2, three concentration of J3 is propped up 150 of packing by 200ul/, and-80 ℃ of preservations are standby.

The repeatability of each 10 parts in each sample being answered pipes according to " the general fluorescence PCR detection reagent kit of swine streptococcus " of the visible 200508001T of table 6 result, 200508002T, three lot numbers of 200508003T detects, same lot number, with the CV value of a detected result all less than 10%, illustrate that this test kit detects simultaneously and has good repeatability with criticizing reagent.

That three batches of test kits of table 6 are criticized is interior, batch between the replica test data

Lot number	Number of times	Sample	Each detected result (Ct value)										Homogeneous detection statistics result in crowd
																1	2	3	4	5	6	7	8	9	10	Average	Standard deviation	The CV value
			2 0 0 5 0 8 0 0 1 T	1	J1	17.67	17.36	17.99	17.54	17.65	17.79	18.36	17.56	18.49	18.28														17.87	0.390	2.18％
J2	20.24	20.35														21.05	20.88	20.46	21.34	20.74	20.36	20.54	20.82	20.68	0.351	1.70％
					J3	23.62	24.54	23.27	24.05	23.79	23.46	23.97	23.56	23.79	23.86												23.79	0.354	1.49％
2	J1	17.68														17.26	18.38	17.67	17.58	17.86	17.67	18.28	17.57	17.40	17.74	0.354				2.00％
				J2	20.56	21.58	20.89	20.86	20.62	20.35	20.55	21.29	20.76	20.72	20.82												0.368	1.77％
	J3	24.38														24.88	23.94	24.03	23.79	23.63	23.53	24.68	23.89	23.57	24.03	0.468			1.95％
				3	J1	17.32	18.23	17.73	17.6	17.74	18.19	17.71	18.04	17.53	17.44												17.75	0.309		1.74％
J2	21.34	20.68														20.84	20.62	20.34	20.37	20.55	20.45	20.57	20.61	20.64	0.288	1.39％
					J3	23.78	23.34	23.57	23.45	24.34	23.68	24.34	23.57	23.34	23.49												23.69	0.369	1.56％

Lot number	Number of times	Sample	Each detected result (Ct value)										Homogeneous detection statistics result in crowd
																1	2	3	4	5	6	7	8	9	10	Average	Standard deviation	The CV value
			2 0 0 5 0 8 0 0 2 T	1	J1	17.68	17.34	17.68	17.45	17.79	17.45	17.56	17.75	17.45	17.55														17.57	0.150	0.85％
J2	20.59	20.8														20.26	20.75	21.22	20.58	20.95	20.26	20.78	20.16	20.64	0.336	1.63％
					J3	23.97	23.45	23.79	23.45	23.67	23.56	23.68	23.45	23.57	23.32												23.59	0.192	0.81％
2	J1	17.35														17.68	17.56	17.49	17.79	17.45	17.56	17.79	17.56	17.62	17.59	0.141				0.80％
				J2	20.44	20.69	20.45	20.79	20.45	20.79	20.48	20.79	20.43	20.63	20.59												0.160	0.78％
	J3	23.69														23.45	23.57	24.24	23.57	23.79	23.34	23.67	23.35	23.61	23.63	0.259			1.10％
				3	J1	17.68	17.34	17.79	17.56	17.49	17.56	17.45	17.8	17.45	17.56												17.57	0.150		0.85％
J2	20.79	20.7														21.25	20.79	20.45	20.29	20.56	20.69	20.86	20.74	20.71	0.257	1.24％

		J3	23.68	23.79	23.8	23.45	23.46	23.68	23.56	23.45	23.79	23.71	23.64	0.145	0.61％
																2 0 0 5 0 8 0 0 3 T	1	J1	17.45	17.68	17.45	17.79	17.45	17.9	17.68	18.08	17.45	17.59	17.65	0.219	1.24％
J2	20.66	20.65	20.49	20.65	20.48	21.45	20.79	20.45	20.79	20.67	20.71	0.287	1.39％
														J3	23.72			23.56	23.78	23.46	23.56	24.34	23.58	24.45	23.68	23.59	23.77	0.342	1.44％
2	J1	17.73	17.52	17.44	17.56	17.26	17.72	18.13	17.65	18.09	17.41	17.65	0.282																	1.60％
														J2	20.64		20.65	20.7	20.56	20.79	20.07	20.45	20.34	20.67	20.9	20.58	0.239	1.16％
	J3	23.56	23.79	23.56	23.76	23.8	23.45	23.69	23.64	23.97	23.73	23.70	0.149																0.63％
														3	J1		17.48	17.34	17.57	17.85	17.38	17.54	17.85	17.58	17.39	17.56	17.55	0.178		1.01％
J2	20.88	20.42	20.85	20.35	20.85	20.54	20.68	20.54	20.58	20.83	20.65	0.194	0.94％
															J3		23.48	23.56	23.76	24.35	24.56	24.34	23.68	23.76	23.65	23.77	23.89	0.379	1.58％

" the universal fluorescence PCR detection reagent kit of swine streptococcus " according to the visible 200508001T of table 7 result, 200508002T, three lot numbers of 200508003T detects three increments repeatability originally, same lot number, the CV value between three detected results illustrates that all less than 10% this test kit also has good repeatability with the detected result of batch reagent, different time separately.

Three batches of test kits of table 7 are criticized interior replica test data separately

The reagent lot number	Detect sample	Detect sample number	Repeated statistics in batch
						Average Ct value	Standard deviation	The CV value
			200508001T	J1
	30	17.44			0.288	1.39％
				J2
	30	20.62			0.257	1.24％
			J3
	30	23.83		0.379	1.58％
			200508002T			J1
	30	17.64		0.309	1.74％
						J2
	30	20.43		0.287	1.39％
			J3
	30	23.83		0.351	1.70％
			200508003T			J1
	30	17.56		0.368	1.77％
						J2
	30	20.62		0.178	1.01％
			J3
	30	23.26		0.259	1.10％

" the universal fluorescence PCR detection reagent kit of swine streptococcus " according to the visible 200508001T of table 8 result, 200508002T, three lot numbers of 200508003T detects three increments repeatability originally, three lot number reagent, the CV value between three detected results illustrates that all less than 10% the detected result of this test kit different batches reagent, different time also has good repeatability separately.

Three batches of test kits of table 8 are criticized a replica test data

The reagent lot number	Detect sample	Detect sample number	Repeated statistics between batch
						Average Ct value	Standard deviation	The CV value
			200508001T 200508002T 200508003T	J1	90				17.23	0.160	0.78％
J2	90	20.32				0.219	1.24％
				J3	90			23.25	0.336	1.63％

According to above testing data and statistical study, that this test kit is criticized is interior, batch between repeated result's CV value all less than 10%, illustrate have well batch in, repeated between criticizing.

Embodiment 5, with comparison (bacterium separation technology) laboratory report of other diagnostic methods

1, material

The bacterial isolates and the source that are applied in the method research process see Table 4.

2, method

1) to the detection of clinical sample-pig brush,throat:

50 parts of the pig brush,throats on random acquisition pig farm, place 1.0ml 0.01M pH7.2 PBS salt solution, after 4 ℃ of immersions of spending the night, get supernatant after the extruding and be divided into 2 parts, a copy of it detects with universal detection method, and portion carries out the bacterium isolation identification with blood agar plate to the swab of taking.

2) to the detection of clinical sample-pig tonsil

108 parts of the pig tonsils in random acquisition slaughterhouse, take by weighing 1g and grind to form 20% suspension with 0.01M pH7.2 PBS salt solution, suspension is divided into 2 parts, and a copy of it detects with universal detection method, and portion carries out the bacterium isolation identification with blood agar plate to the tonsilla tissue suspension.

3, result

1) to the detected result of clinical sample brush,throat:

As shown in Figure 4, when 50 parts of pig brush,throats are detected with the universal detection method of setting up, detect 1 part of positive, the result of blood agar separation test is all negative.

2) to the detected result of clinical sample-pig tonsil suspension:

As shown in Figure 5, to 108 parts of pig tonsil suspensions of random acquisition, detect with the universal detection method of setting up, the result is all negative.The result of blood agar separation test is also all negative.

In this research, we tentatively are used for the method for setting up the detection of clinical sample, sample is all from certain healthy pig farm random acquisition, in detection to brush,throat, detect a positive with universal method, but the result of bacterium separation test is all negative, and the 33# sample fails to be separated to bacterium may be long relevant with the sample shelf-time; To 108 parts of pig tonsil sample detection the time, the detected result of fluorescence PCR method is all negative, and is in full accord with the result of bacterium separation test.

Sequence table

＜110〉People's Republic of China Beijing Entry-Exit Inspection and Quarantine Bureau

China Veterinery Drug Inspection Office

＜120〉nucleotide sequence, general detection kit and the method for detection swine streptococcus

<130>

<160>4

<170>PatentIn version 3.2

<210>1

<211>20

<212>DNA

＜213〉synthetic

<400>1

taacggctca ccaaggcttc 20

<210>2

<211>20

<212>DNA

＜213〉synthetic

<400>2

ttgccgaaga ttccctactg 20

<210>3

<211>24

<212>DNA

＜213〉synthetic

<400>3

atacatagcc gacctgagag ggtg 24

<210>4

<211>114

<212>DNA

＜213〉swine streptococcus

<400>4

taacggctca ccaaggcatc gatacatagc cgacctgaga gggtgatcgg ccacactggg 60

actgagacac ggcccagact cctacgggag gcagcagtag ggaatcttcg gcaa 114

Claims (6)

1. one group of nucleotide sequence that detects swine streptococcus, as follows:

1)SEQ ID NO：1：5’-TAA CGG CTC ACC AAG GCT TC-3’

2)SEQ ID NO：2：5’-TTG CCG AAG ATT CCC TAC TG-3’

3)SEQ ID NO：3：5’-ATA CAT AGC CGA CCT GAG AGG GTG-3’。

2. the nucleotide sequence of detection swine streptococcus according to claim 1 is characterized in that: 5 ' end mark report fluorophor FAM sequence 3), 3 ' end mark cancellation fluorophor TAMRA.

3. common reagent box that detects swine streptococcus, the 48tests/ box, composed of the following components:

1) DNA extraction reagent: 5ml/ pipe * 6 pipes;

2) PCR reaction solution, 750 μ L * 1 pipe comprise: 1 * PCR damping fluid, 3.0mM MgCL ₂, 0.2mMdNTP, 0.3 μ mol/L primer, 1,0.3 μ mol/L primer 2 and 0.1 μ mol/L probe; Wherein, the nucleotide sequence of primer 1 is shown in sequence table SEQ ID NO:1, the nucleotide sequence of primer 2 is shown in sequence table SEQ ID NO:2, the nucleotide sequence of probe is shown in sequence table SEQ ID NO:3,5 ' end mark report fluorophor FAM of probe, 3 ' end mark cancellation fluorophor TAMRA;

3) Taq archaeal dna polymerase 5U/ μ L, 15 μ L * 1 pipe;

4) the sterilization purified water of no DNA enzyme, 1mL * 1 pipe;

5) negative control: 1mL * 1 pipe: the aseptic SPF pig substantial viscera of winning, make 20% suspension with 0.01mol/L pH7.2PBS buffer saline, 60 ℃ of effects deactivation in 1 hour;

6) positive control: 1mL * 1 pipe, for containing the recombinant plasmid of swine streptococcus 16S rDNA specific fragment, the Ct value is 22.0～28.0.

4. a kind of common reagent box that detects swine streptococcus according to claim 3, it is characterized in that: the preparation method of described positive control is: adopt the primer sequence shown in SEQ ID NO:1 and the SEQ ID NO:2 that swine streptococcus is carried out the PCR reaction, reclaim pcr amplification product, obtain the specific fragment of 114bp, be connected with PGEM-T easy carrier after reclaiming purifying, transform the JM109 competent cell, alkaline lysis method of extracting plasmid DNA, cut through PCR and enzyme and to identify the positive recombinant plasmid of back acquisition, 10 times of serial dilution lysates are measured the Ct value and are 22.0～28.0 and promptly can be used as positive control.

5. a kind of common reagent box that detects swine streptococcus according to claim 3 is characterized in that: the nucleotide sequence of described swine streptococcus 16S rDNA specific fragment is shown in sequence table SEQ ID NO:4.

6. a method that detects swine streptococcus comprises the steps:

1) extracts sample DNA;

2) sample DNA that extracts is carried out pcr amplification:

Primer sequence is 5 '-TAA CGG CTC ACC AAG GCT TC-3 '

5’-TTG CCG AAG ATT CCC TAC TG-3’

Fluorescent probe is 5 '-[FAM] ATA CAT AGC CGA CCT GAG AGG GTG[TAMRA]-3 ';

Amplification condition is 92 ℃/3min; 92 ℃/5sec, 60 ℃/30sec, fluorescence is collected in 45 circulations during each loop ends;

3) whether the fluorescence intensity of mensuration PCR reaction system exists swine streptococcus in the judgement sample; The result describes and judges, feminine gender, and no Ct value and do not have amplification curve, showing does not have swine streptococcus in the sample; The positive, Ct value≤30.0, and specific amplification curve appears, there is swine streptococcus in the expression sample.

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