CN102174653A - Haemophilus parasuis real-time fluorescent quantitative PCR (polymerase chain reaction) detection method - Google Patents

Abstract

The invention discloses a haemophilus parasuis real-time fluorescent quantitative PCR (polymerase chain reaction) detection method which selects the 16S rRNA gene (AB004028) which has high conservation and specificity and is the main target gene of bacteria classification and determination as the amplification target gene, takes a high-conservation area of the gene as an amplification area to design a specificity primer, and establishes a haemophilus parasuis real-time fluorescent quantitative PCR detection method embedding a fluorescent dye SYBR Green I and DNA, wherein the DNA of the initial template of the haemophilus parasuis 16S rRNA gene can be accurately quantified according to the standard curve of the known standard product, and equated to the number of bacteria. The method disclosed by the invention has strong generality and high specificity, can detect many HPS serotypes, is free from influence of related bacteria, and can be applied to quick and accurate quantitative detection of the haemophilus parasuis seriously impairing development of the pig industry.

Description

The haemophilus parasuis real-time fluorescence quantitative PCR detection method

Technical field

The present invention relates to fluorescence quantitative PCR detection, especially a kind of haemophilus parasuis real-time fluorescence quantitative PCR detection method.

Background technology

(Haemophilus parasuis HPS) belongs to Pasteur's moral Cordycepps hemophilus to haemophilus parasuis.The common field planting of HPS is at the pig upper respiratory tract, do not cause the pig morbidity under the normal condition, but when being in stress situation or lower immune function, body can break through the defense mechanism of the upper respiratory tract, invade a plurality of organs of body, cause polyserositis, sacroiliitis, severe patient causes fervescence, expiratory dyspnea even death, brings enormous economic loss to pig industry.

Diagnosis to HPS mainly is bacterium separation and Culture and conventional PCR method at present.The culture condition of HPS requires high, needs NAD, horse serum and 5%CO ₂Environment, incubation time needs 24～48h, usually occurs living contaminants in the separation and Culture process.General PCR method (the OLIVEIRA S that sets up such as the Oliverira of Shi Yonging, GALINA L, PIJOAN C.Development of a PCR test to diagnose Haemophilus parasuis infections[J] .J Vet Diagn Invest, 2001, though 13:1495-1501) can directly detect pathological material of disease, during check fee and can not be accurately quantitatively and exist false-positive may.

Real-time fluorescence PCR is a kind of round pcr that DNA quantity is carried out check and analysis, have easy and simple to handle, susceptibility and advantages such as high specificity, good reproducibility, and the result has real-time, and is more accurate and visual, be widely used in that expression of gene detects and quantitative examination in.People such as Yu Jiang point out that the 16SrRNA gene order should not be used as fluorescence quantitative PCR detection, and set up with the infB gene as the HPS fluorescence quantitative PCR detection technique of target gene (in the river, Wu Jia is strong etc. the foundation of haemophilus parasuis fluorescence quantitative PCR detection technique and application. and ecology of domestic animals is reported .2010, and 31 (4): 77-81).But this research only limits to HPS serum 5 types, and the versatility of other serotype is not done effective checking, and its method can only distinguish haemophilus parasuis and suis and pasteurellosis bacillus, and decorrelation bacterium interference performance is limited.

Summary of the invention

The technical problem to be solved in the present invention provide a kind of fast, sensitive, accurately, versatility is good, immunity from interference is strong haemophilus parasuis real-time fluorescence quantitative PCR detection method.

Adopt following technical scheme for solving the problems of the technologies described above the present invention: the haemophilus parasuis real-time fluorescence quantitative PCR detection method, promptly detect by the fluorescent signal that is sent in the chimeric PCR reaction of fluorescence dye, 16S rRNA gene order with the haemophilus parasuis of accession number AB004028 is reference, and its primer sequence and extension increasing sequence are respectively:

Primer sequence 15 '-GAC GGG AAA CTG TCG CTA-3 '

Primer sequence 25 '-CTA GAG ATC GTC GGC TTG-3 '

Extension increasing sequence is the base sequence of sequence table SEQ .ID.No.3.

Aforesaid method comprises the steps:

＜1〉preparation dna sample: get the extraction purifying that sample to be checked carries out total DNA;

＜2〉real-time fluorescence quantitative PCR reaction: get step＜1〉the gained dna sample carries out the fluorescent PCR amplification as template, and carry out the fluorescent PCR amplification with standard substance DNA and the positive check sample of deionized water and negative control sample respectively simultaneously;

＜3〉set up typical curve: the standard substance DNA that gets concentration known is diluted to 5 * 10 by 10 times of dilution methods ⁷～5 * 10 ⁰8 concentration gradients of copy/μ L,〉PCR reaction system and response procedures increases set by step＜2, after reaction finishes, according to the cycle threshold Ct drafting quantitative fluorescent PCR typical curve of each concentration gradient that obtains;

Cycle threshold Ct and typical curve contrast with dna sample obtain the 16S rRNA gene fragment copy concentrations in the dna sample to be checked, obtain the bacterial number of the haemophilus parasuis in the sample in view of the above.

The fusion program of reaction system, amplification program and the mensuration amplified fragments of quantitative fluorescent PCR reaction is respectively:

Reaction system: PCR mixed solution 20 μ L, wherein each 0.3 μ L, dna profiling 2.5 μ L and deionized water 7.9 μ L of the upstream and downstream primer of SYBR GreenI Mix 9 μ L, 5pmol/ μ L;

Amplification program: 95 ℃ of 5min, carry out 40 circulations subsequently, each circulation comprises 95 ℃ of 20s, 62 ℃ of 20s, 68 ℃ of 20s;

Measure the fusion program of amplified fragments: rise to 95 ℃ by the temperature rise rate of per 0.5 ℃/10s from 55 ℃ and carry out the melting curve analysis verification.

The characteristics that the present invention is more according to HPS serotype, choose have high conservative and specificity and be division bacteria identify main target gene 16S rRNA gene (GENBANK accession number AB004028) as the amplified target gene, belong to the inner height conservative region with this gene and design Auele Specific Primer, set up fluorescence dye SYBR Green I and the chimeric haemophilus parasuis real-time fluorescence quantitative PCR detection method of DNA as amplification region.Because fluorescence signal intensity is directly proportional with the quantity of amplified fragments, typical curve by the known standard product can carry out the DNA of haemophilus suis 16S rRNA gene original template accurately quantitatively, and the section of DNA sequence on this gene line bacterial chromosome, bacterial chromosome is a monoploid, so the copy number of 16S rRNA gene can be equal to number of bacteria.In addition, compare with conventional PCR, the nucleic acid amplification of real-time fluorescence PCR and detection are all carried out in same pipe, judge by fluorescent signal whether amplified fragments increases, need not electrophoresis detection, solved the pollution problem of PCR false positive and nucleic acid dye ethidium bromide environment.At last, present method highly versatile, specificity height can detect multiple HPS serotype, and don't are subjected to streptococcus suis 2-type, streptococcus aureus, intestinal bacteria DH _{5 α}Influence with relevant bacterium such as moscow' typhisuises.In sum, the present invention can be used for rapidly and accurately the haemophilus parasuis of serious harm pig industry development is carried out detection by quantitative.

Description of drawings

Fig. 1 is the qualitative PCR electrophorogram of the primer of the present invention, and among the figure: 1 is molecular weight marker DNA Marker 1 (600bp); 2 is HPS serum 4 types; 3 is HPS serum 5 types; 4 is HPS serum 9 types; 5 is HPS serum 14 types.

Fig. 2 is a melting curve analysis chart of the present invention.

Fig. 3 is a pcr amplification dynamic curve of the present invention, and among the figure: 1 is 5 * 10 ⁷Copy/μ L; 2 is 5 * 10 ⁶Copy/μ L; 3 is 5 * 10 ⁵Copy/μ L; 4 is 5 * 10 ⁴Copy/μ L; 5 is 5 * 10 ³Copy/μ L; 6 is 5 * 10 ²Copy/μ L; 7 is 5 * 10 ¹Copy/μ L.

Fig. 4 is a canonical plotting of the present invention.

Fig. 5 is a specificity test chart of the present invention, and among the figure: 1 is HPS serum 5 types; 2 is HPS serum 9 types; 3 is HPS serum 14 types; 4 is HPS serum 4 types; 5 is other bacterium: streptococcus suis 2-type, streptococcus aureus, intestinal bacteria DH _{5 α}, moscow' typhisuis.

Fig. 6 is replica test figure of the present invention, and among the figure: 1 is 10 ⁷Copy/μ L; 2 is 10 ⁵Copy/μ L; 3 is 10 ³Copy/μ L.

Fig. 7 is the schema of haemophilus parasuis real-time fluorescence quantitative PCR detection method of the present invention.

Fig. 8 is the clinical detection figure as a result of real-time fluorescence PCR of the present invention, and among the figure: 1 is that pericardial fluid 1,2 is that pericardial fluid 2,3 is that pericardial fluid 3,4 is that pericardial fluid 4,5 is that pericardial fluid 5,6 is that synovial fluid 1,7 is that synovial fluid 2,8 is that synovial fluid 3,9 is that synovial fluid 4,10 is a synovial fluid 5.

Embodiment

The research of haemophilus parasuis real-time fluorescence quantitative PCR detection method

1 materials and methods

1.1 key instrument and reagent

ICycler iQ5 quantitative real time PCR Instrument is available from U.S. Bio-Rad company; Conventional PCR instrument is available from Japanese TaKaRa company; Gel imaging system is available from U.S. Alpha Inotech company; ND-1000 trace ultraviolet spectrophotometer is available from U.S. Thermo company.RealMasterMix PCR kit for fluorescence quantitative (SYBR), bacterial genomes DNA extraction test kit, sepharose DNA reclaim test kit, a small amount of plasmid extraction kit available from TIANGEN Biotech (Beijing) Co., Ltd.; PCR Taq Mix, DNA Marker 1 contain bio tech ltd available from east, Guangdong; The PMD-18T carrier is available from precious biotechnology (Dalian) company limited; The TSA nutrient agar is available from Beijing overpass Technical responsibilities company limited; Horse serum is available from U.S. Hylone company; NAD is available from Beijing Suo Laibao Science and Technology Ltd..

1.2 bacterial strain

HPS serum 4 types, 5 types, 9 types and 14 types; Streptococcus suis 2-type, streptococcus aureus, intestinal bacteria DH _{5 α}, moscow' typhisuis preserved by this laboratory.

1.3 design of primers

16S rRNA gene order (AB004028) with HPS is reference, using primer-design software DNA STAR and Oligo6.0 analyzes the 16S rRNA gene of various bacteria, select the 16S rRNA gene of HPS to belong to interior conserved regions as amplification region, design a pair of primer, amplified fragments (seeing sequence table SEQ .ID.No.3) length is 136bp.Primer sequence is 5 '-GAC GGG AAA CTG TCG CTA-3 ' (seeing sequence table SEQ .ID.No.1) and 5 '-CTA GAG ATC GTCGGC TTG-3 ' (seeing sequence table SEQ .ID.No.2), primer is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd, concrete parameter of primer such as table 1.

Table 1 primer parameter list

1.4 the preparation of template DNA

The single bacterium colony of picking HPS is rule on the TSA nutrient agar that contains 5% horse serum and 0.4mg/mL NAD, places 5%CO ₂Cultivate 36h for 37 ℃ in the environment, physiological saline wash-out bacterium, with bacterial genomes DNA extraction test kit extracting DNA of bacteria, the extracting method by specification carries out.

1.5 qualitative PCR amplification

Adopt the HPS primer of design that extractive dna profiling is carried out pcr amplification, the PCR reaction system is 50 μ L, comprises PCR Taq Mix 12 μ L, each 1 μ L of upstream and downstream primer (25pmol/ μ L), template DNA 5 μ L, ultrapure water 31 μ L.Reaction conditions is 95 ℃ of pre-sex change 5min; 95 ℃ of 20S of 35 round-robin, 60 ℃ of 20S, 72 ℃ of 20S, last 72 ℃ are extended 5min.

1.6 the preparation of standard positive plasmid

The PCR product reclaims test kit with sepharose DNA and reclaims the PCR product behind 2% agarose gel electrophoresis.The PCR product spends the night with the PMD-18T carrier and is connected back transformed into escherichia coli DH _{5 α}Extract to transform the bacterium colony plasmid that grows with a small amount of plasmid extraction kit, positive plasmid is served the order-checking of extra large Ying Jun Bioisystech Co., Ltd after PCR and double digestion are identified.

After the correct positive plasmid of order-checking measured purity with ultraviolet spectrophotometer, copy number=(plasmid concentration/plasmid molecule quality) * A Fodeluo constant (6.02 * 10 by formula ²³) calculate the copy number of plasmid.With 5 * 10 ⁹The plasmid of copy/μ L carries out 10 times of gradient dilutions, respectively with 5 * 10 ⁷～5 * 10 ⁰The plasmid of copy/μ L is used for real-time fluorescence PCR as standard substance.

1.7 the foundation of real time fluorescent PCR method

Adopt chessboard method screening primer concentration (2.5～25pmol/ μ L) and PCR annealing temperature (58～62 ℃), optimize the real-time fluorescence PCR The optimum reaction conditions in the cycle threshold (Ct) of minimum, to obtain maximum fluorescent signal.After real-time fluorescence PCR finishes, measure light absorption value from 55～95 ℃ by per 0.5 ℃/10s and carry out the melting curve analysis, determine whether PCR is specific amplification.

1.8 the foundation of typical curve

Be 5 * 10 with concentration respectively ⁷～5 * 10 ⁰The plasmid standard of copy/μ L is a template, adopts optimized reaction conditions to carry out real-time fluorescence PCR.Ct threshold setting principle is for surpassing the vertex of negative control amplification curve (random noise line), and Ct value＞35 are negative, and Ct value＜35 are positive.The Ct value that quantitative real time PCR Instrument can record according to each concentration standard product is an X-coordinate with the logarithm of standard substance copy number, is that ordinate zou is set up typical curve with the Ct value.

1.9 the specificity of real time fluorescent PCR method and replica test

Extract HPS serum 4 types, 5 types, 9 types and 14 types respectively, streptococcus suis 2-type, streptococcus aureus, intestinal bacteria DH _{5 α}, moscow' typhisuis DNA carry out real-time fluorescence PCR, with the specificity of evaluation method.

With the method for setting up respectively to 10 ³, 10 ⁵, 10 ⁷The plasmid of copy/μ L detects, and each concentration sample repeats 3 times, carries out the calculating of the variation coefficient according to the Ct of each concentration, with the repeatability of evaluation method.

2 results

2.1 segmental clone of purpose and evaluation

As shown in Figure 1, the HPS primer with design carries out pcr amplification to the DNA of HPS serum 4 types, 5 types, 9 types and 14 types respectively, appearance and expection amplification length identical segments behind the PCR product electrophoresis.The PCR product is after sepharose reclaims, and spending the night with the PMD-18T carrier is connected also transformed into escherichia coli DH _{5 α}The positive plasmid that obtains is served the order-checking of extra large Ying Jun Bioisystech Co., Ltd after PCR and double digestion are identified, sequencing result and reference sequences are in full accord.

2.2 the foundation of real time fluorescent PCR method

Adopt chessboard method that PCR annealing temperature and primer concentration are optimized, obtained real-time fluorescence PCR optimum response system and condition.The total reaction system is 20 μ L, wherein RealMasterMix 9 μ L, upstream and downstream primer (5pmol/ μ L) each 0.3 μ L, dna profiling 2.5 μ L and ultrapure water 7.9 μ L.Reaction conditions is: 95 ℃ of pre-sex change 5min; Carry out 40 95 ℃ of 20s subsequently, 62 ℃ of 20s, the circulation of 68 ℃ of 20s.

As shown in Figure 2, carry out the melting curve analysis behind the real-time fluorescence PCR to determine whether reaction is specific amplification.Melting curve only forms a specific peak, and Tm is 84.5 ℃, shows that the amplification and the primer dimer that do not have non-specific product form, and the PCR reaction conditions has obtained good optimization, and data can be used for quantitative conversion.

2.3 the foundation of typical curve

The extracting positive plasmid is surveyed its OD ₂₆₀/ OD ₂₈₀Value is 1.93.Calculate the positive plasmid copy number, with 5 * 10 ⁹The plasmid of copy/μ L carries out 10 times of gradient dilutions as standard substance, adopt optimized reaction conditions to carry out the real-time fluorescence PCR amplification, the Ct value that quantitative real time PCR Instrument records according to each standard substance generates the amplification dynamic curve (see figure 3) and the typical curve (see figure 4) of real-time fluorescence PCR automatically.

The amplification dynamic curve shows, when template concentrations is 5 * 10 ¹During copy/μ L, the amplified fluorescence curve is arranged; And template concentrations is 5 * 10 ⁰During copy/μ L, do not have amplification curve, show that it is 5 * 10 that this method detects lowest limit ¹Copy/μ L.The typical curve formula is Ct=-3.410 * log copy number+25.346, and the slope of typical curve is-3.410, and intercept is 25.346, relation conefficient (R ²) be 0.998.By read the Ct value of sample to be tested from instrument, substitution typical curve formula can converse its initial bacterium copy number.

2.4 specificity test

With HPS serum 4 types, 5 types, 9 types and 14 types, streptococcus suis 2-type, streptococcus aureus, intestinal bacteria DH _{5 α}, moscow' typhisuis DNA be that template is carried out real-time fluorescence PCR, as shown in Figure 5, amplification curve diagram shows to have only the detection of HPS serum 4 types, 5 types, 9 types and 14 types to reach the Ct threshold value, and other bacterium does not have amplification curve, shows that the real-time fluorescence PCR of foundation has higher specificity.

2.5 replica test

With the method for setting up respectively to 10 ³, 10 ⁵, 10 ⁷The plasmid of copy/μ L detects, and each concentration sample repeats 3 times.As shown in Figure 6, amplification curve diagram shows that the amplification curve of each concentration sample is almost overlapping, and error is very little, and the variation coefficient of each concentration sample repeatability shows that all less than 2% the real-time quantitative PCR of foundation has better repeatability and stability as calculated.

The detection of embodiment clinical sample

Detect with pericardial fluid, the synovial fluid of the haemophilus parasuis real-time fluorescence quantitative PCR detection method of setting up (method A), and compare with bacterium separation and Culture (method B) and conventional PCR (method C) to 10 parts of doubtful HPS infection of clinical collection.

As shown in Figure 7, haemophilus parasuis real-time fluorescence quantitative PCR detection method concrete steps are as follows:

＜1〉preparation dna sample: get the extraction purifying that sample to be checked carries out total DNA;

Pericardial fluid is handled: draw 10 microlitre pericardial fluids and join in the EP pipe that 100 microlitre ultrapure waters are housed, boil 10min, the centrifugal 5min of 12000rpm gets supernatant 3 microlitres as dna profiling.

Synovial fluid is handled: draw 10 microlitre synovial fluids and join in the EP pipe that 100 microlitre ultrapure waters are housed, boil 10min, the centrifugal 5min of 12000rpm gets supernatant 3 microlitres as dna profiling.

＜2〉real-time fluorescence quantitative PCR reaction: get step＜1〉the gained dna sample carries out the fluorescent PCR amplification as template on the real-time fluorescence quantitative PCR instrument, and carry out the fluorescent PCR amplification with standard substance DNA and the positive check sample of deionized water and negative control sample respectively simultaneously, wherein the fusion program of reaction system, amplification program and the mensuration amplified fragments of quantitative fluorescent PCR reaction is respectively:

Reaction system: PCR mixed solution 20 μ L, wherein each 0.3 μ L, dna profiling 2.5 μ L and deionized water 7.9 μ L of the upstream and downstream primer of SYBR Green I Mix 9 μ L, 5pmol/ μ L;

Amplification program: 95 ℃ of 5min, carry out 40 circulations subsequently, each circulation comprises 95 ℃ of 20s, 62 ℃ of 20s, 68 ℃ of 20s;

Measure the fusion program of amplified fragments: rise to 95 ℃ by the temperature rise rate of per 0.5 ℃/10s from 55 ℃ and carry out the melting curve analysis verification.

＜3〉set up typical curve: the standard substance DNA that gets concentration known is diluted to 5 * 10 by 10 times of dilution methods ⁷～5 * 10 ⁰8 concentration gradients of copy/μ L, set by step＜2〉PCR reaction system and response procedures increase on the real-time fluorescence quantitative PCR instrument, after reaction finishes, according to the cycle threshold Ct employing computer automatic drafting quantitative fluorescent PCR typical curve (as Fig. 4) of each concentration gradient that obtains;

Cycle threshold Ct (as Fig. 8) and typical curve contrast with dna sample obtain the 16S rRNA gene fragment copy concentrations in the dna sample to be checked, obtain the bacterial number of the haemophilus parasuis in the sample in view of the above.

Adopt the clinical sample detected result such as the table 2 of three kinds of methods:

Three kinds of HPS detection methods of table 2 relatively

The result shows, three kinds of detection method detected result unanimities, and promptly all clinical samples all are the HPS positive.Fluorescence quantifying PCR method and bacterial plate counts are surveyed the bacterial number basically identical, and conventional P CR method can not be quantitative.

Above-mentioned studies show that, present method is all positive to the detected result of HPS strong virus force (5 types and 14 types), mesogenic (4 type) and low virulence (9 type) bacterial strain, has confirmed the versatility of method on HPS serotype detects.In addition, present method also has higher specificity, can only detect HPS, and can not detect streptococcus suis 2-type, streptococcus aureus, intestinal bacteria DH _{5 α}And moscow' typhisuis.Simultaneously, the typical curve that this research is set up according to 16S rRNA gene plasmid, linearity range is wide, 5 * 10 ⁷～5 * 10 ¹Present good linear relationship during copy/μ L, relation conefficient (R ²) be 0.998, the minimum detection limit reaches 5 * 10 ¹Copy/μ L.The replica test substantive approach has higher repeatability and stability, and the variation coefficient is all less than 2%.It is consistent with bacterium separation and Culture and conventional PCR qualification result that present method detects clinical sample, but operation is simpler and quick, can obtain detected result in 1.5h.Therefore, HPS real-time fluorescence quantitative PCR detection method of the present invention has fast, sensitive, accurate and stable characteristics, can be used for the clinical rapid detection that HPS infects.

Claims (3)

1. haemophilus parasuis real-time fluorescence quantitative PCR detection method, detect by the fluorescent signal that is sent in the chimeric PCR reaction of fluorescence dye, it is characterized in that the 16S rRNA gene order with the haemophilus parasuis of accession number AB004028 is reference, its primer sequence and extension increasing sequence are respectively:

Primer sequence 15 '-GAC GGG AAA CTG TCG CTA-3 '

Primer sequence 25 '-CTA GAG ATC GTC GGC TTG-3 '

Extension increasing sequence is the base sequence of sequence table SEQ .ID.No.3.

2. haemophilus parasuis real-time fluorescence quantitative PCR detection method according to claim 1 is characterized in that described method comprises the steps:

＜1〉preparation dna sample: get the extraction purifying that sample to be checked carries out total DNA;

＜2〉real-time fluorescence quantitative PCR reaction: get step＜1〉the gained dna sample carries out the fluorescent PCR amplification as template, and carry out the fluorescent PCR amplification with standard substance DNA and the positive check sample of deionized water and negative control sample respectively simultaneously;

＜3〉set up typical curve: the standard substance DNA that gets concentration known is diluted to 5 * 10 by 10 times of dilution methods ⁷～5 * 10 ⁰8 concentration gradients of copy/μ L,〉PCR reaction system and response procedures increases set by step＜2, after reaction finishes, according to the cycle threshold Ct drafting quantitative fluorescent PCR typical curve of each concentration gradient that obtains;

Cycle threshold Ct and typical curve contrast with dna sample obtain the 16S rRNA gene fragment copy concentrations in the dna sample to be checked, obtain the bacterial number of the haemophilus parasuis in the sample in view of the above.

3. haemophilus parasuis real-time fluorescence quantitative PCR detection method according to claim 2 is characterized in that the fusion program of reaction system, amplification program and the mensuration amplified fragments of described quantitative fluorescent PCR reaction is respectively:

Reaction system: PCR mixed solution 20 μ L, wherein each 0.3 μ L, dna profiling 2.5 μ L and deionized water 7.9 μ L of the upstream and downstream primer of SYBR Green I Mix 9 μ L, 5pmol/ μ L;

Amplification program: 95 ℃ of 5min, carry out 40 circulations subsequently, each circulation comprises 95 ℃ of 20s, 62 ℃ of 20s, 68 ℃ of 20s:

Measure the fusion program of amplified fragments: rise to 95 ℃ by the temperature rise rate of per 0.5 ℃/10s from 55 ℃ and carry out the melting curve analysis verification.

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