CN102134598B - Method for detecting Aeromonas hydrophila by FQ-PCR (fluorescent quantitative polymerase chain reaction) - Google Patents

Abstract

The invention provides a kit for detecting Aeromonas hydrophila by FQ-PCR (fluorescent quantitative polymerase chain reaction). In a reagent packed in the kit, a sense primer and a reverse primer and TaqMan probe have one or two groups of A group and B group. Compared with the prior art, as the based factors only exist in the virulent Aeromonas hydrophila strain, the method can accurately detect whether the virulent Aeromonas hydrophila strain exists in the sample, thereby improving the early warning accuracy of the disease. Simultaneously, if the detection result shows that the template contains the hemolysin gene (hlyA) or serine proteinase gene (ahpA) by using the A group or B group reagent, this indicates that the disease has certain pathogenicity; and if the detection result shows that the template contains the hemolysin gene (hlyA) and serine proteinase gene (ahpA) by using the two groups of reagents, this indicates that the disease has high pathogenicity.

Description

Detect the method for Aeromonas hydrophila with FQ-PCR

Technical field

What the present invention relates to is a kind of method that detects Aeromonas hydrophila with FQ-PCR, belongs to the mensuration or the method for inspection that comprise enzyme or microorganism in the chemistry.

Background technology

(Aeromonas hydrophila) is widely distributed at occurring in nature for Aeromonas hydrophila, be prevalent in fresh water, sewage, mud, soil and the human feces, can cause all kinds of zoogenetic infections, and symptoms such as the people can poison by food when infecting Aeromonas hydrophila, diarrhoea, and being immunosuppressed patient and liver function disease patient's opportunistic pathogen, is a kind of typical people, animal, fish ill pathogenic bacteria altogether.Aeromonas hydrophila is except bringing the tremendous economic loss to culture fishery, it is to public health, and especially the influence aspect clinical disease and food safety is also very serious.

The detection method of traditional Aeromonas hydrophila is that Physiology and biochemistry is identified and immunology diagnosis, but because many biochemical character instabilities of this bacterium, serotype is also complicated, so easily produce erroneous judgement.The report of the PCR detection method aspect of therefore existing multinomial Aeromonas hydrophila, as Chinese invention patent application " aquatic animal and human pathogenic bacteria---Aeromonas hydrophila gene diagnosis kit and detection method (publication number CN1560275) ", Chinese invention patent application " laboratory animal swordtail pathogenic bacteria aeromonas hydrophila PCR detection kit and detection method (publication number CN1978665) ", Chinese invention patent application " PCR detection kit of Aeromonas hydrophila and detection method thereof (publication number CN1506468) ", Chinese invention patent application " a kind of Aeromonas and Aeromonas hydrophila double PCR quick detection kit and detection method (publication number CN101736073A) " and Chinese invention patent application " pathogenic hydrophila gingivalis detects with reagent and detection method (publication number CN101418335) thereof " etc., but above-mentioned PCR detection method all about Aeromonas hydrophila all is a common PCR to be detected, and all can not quantitative assay for qualitative test.

The relevant report that adopts PQ-PCR to detect the Aeromonas hydrophila method is arranged again, as " foundation of Aeromonas hydrophila TaqMan real time PCR detection method " (Wang Haibo etc., " Chinese Journal of Preventive Medicine ", 43 (7), 611-614) disclosed.The specific specificity gene of directed toward bacteria adopts round pcr to carry out the focus that rapid detection is pathogenic bacteria detection in recent years, compare with conventional PCR, but the real-time fluorescence quantitative PCR detection technique has that specificity is stronger, susceptibility is higher, qualitative, quantitative simple and efficient to handle and can effectively solve advantages such as conventional PCR pollution problem.But the object of the design of primers in the disclosure technology is gas lysin gene (aerolysin) and the adhesin gene (adhesins) of Aeromonas hydrophila.

Studies show that in a large number, Aeromonas hydrophila has the branch of pathogenic strains and non-pathogenic bacteria strain, although Aeromonas hydrophila and extracellular products thereof have multiple virulence factor, comprise: adhesin (adhesins), invasion (invasions), enterotoxin (enterotoxins), hemolysin (hlyA), gas lysin (aerolysin), outer membrane protein (outermembrane proteins), serine protease gene (ahpA) etc., these virulence factors play an important role in the pathogenic course of bacterium, but these virulence factors are collaborative mutually in the performance to the body pathogenic effects, the existence of some virulence factor might not be caused a disease and be needed the collaborative of multiple virulence factor just can cause a disease, and some virulence factor just is morbific as long as exist certainly.Wherein hemolysin gene (hlyA) and serine protease gene (ahpA) are exactly to have only morbific Aeromonas hydrophila bacterial strain to possess, be that Aeromonas hydrophila avirulence bacterial strain does not generally have hemolysin gene (hlyA) and serine protease gene (ahpA), therefore measure hemolysin gene (hlyA) and serine protease gene (ahpA) thus the Aeromonas hydrophila of distinguishing virulence is had bigger using value aspect emergency advance warning and the burst disease processing.

Summary of the invention

At above-mentioned deficiency, technical problem to be solved by this invention is to propose a kind of method with FQ-PCR detection Aeromonas hydrophila at the DNA that exists only in virulent strain.

One of method that detects Aeromonas hydrophila with FQ-PCR provided by the invention, prepare the Aeromonas hydrophila template DNA earlier, carry out amplified reaction again, being about to ready amplification reaction solution adds in the template DNA, on the PCR instrument, carry out amplified reaction then and obtain data calculating for computer, last judged result, comprise in the said amplification reaction solution on the factor in the Aeromonas hydrophila bacterial strain that exists only in virulence, downstream primer, wherein the dna sequence dna of upstream primer is 5 '-ACC GCA AGA TCA ACG ATA CCG AGT-3 ', and the dna sequence dna of downstream primer is 5 '-ATC CAG CGA GAT CCG CAC TAT CTT-3 '.

Also comprise the TaqMan probe in the said amplification reaction solution, the dna sequence dna of this TaqMan probe is 5 '-(FAM)-AAC ATC TCG CTG GTT TAC CGG GTC AA-(TAMRA)-3 '.

Two of the method that detects Aeromonas hydrophila with FQ-PCR provided by the invention, prepare the Aeromonas hydrophila template DNA earlier, carry out amplified reaction again, being about to ready amplification reaction solution adds in the template DNA, on the PCR instrument, carry out amplified reaction then and obtain data calculating for computer, last judged result, comprise in the said amplification reaction solution on the factor in the Aeromonas hydrophila bacterial strain that exists only in virulence, downstream primer, wherein the dna sequence dna of upstream primer is 5 '-ACC CAG GAC GGT GCG ATA TAA GAT-3 ', and the dna sequence dna of downstream primer is 5 '-TCT CAC TTG CCT GAA CTG AAG CGA-3 '.

Also comprise the TaqMan probe in the said amplification reaction solution, the dna sequence dna of this TaqMan probe is 5 '-(FAM)-AAC ATC GTT AGC GTT GGC AAT CTC GG-(TAMRA)-3 '.

Three of the method that detects Aeromonas hydrophila with FQ-PCR provided by the invention, prepare the Aeromonas hydrophila template DNA earlier, carry out amplified reaction again, being about to ready amplification reaction solution adds in the template DNA, on the PCR instrument, carry out amplified reaction then and obtain data calculating for computer, last judged result, whole steps is carried out simultaneously with two groups of parallel experiments, comprise in one of them said amplification reaction solution that dna sequence dna is the upstream primer of 5 '-ACC GCA AGA TCA ACG ATA CCG AGT-3 ', dna sequence dna is the downstream primer of 5 '-ATC CAG CGA GAT CCG CAC TAT CTT-3 ', and dna sequence dna is the TaqMan probe of 5 '-(FAM)-AAC ATC TCG CTG GTT TAC CGG GTC AA-(TAMRA)-3 '; Comprise in the two said amplification reaction solutions wherein that dna sequence dna is the upstream primer of 5 '-ACC CAG GAC GGT GCG ATA TAA GAT-3 ', dna sequence dna is the downstream primer of 5 '-TCT CAC TTG CCT GAA CTG AAG CGA-3 ', and dna sequence dna is the TaqMan probe of 5 '-(FAM)-AAC ATC GTT AGC GTT GGC AAT CTC GG-(TAMRA)-3 '.

The method that detects Aeromonas hydrophila with FQ-PCR provided by the invention, use the FQ-PCR method and the Aeromonas hydrophila that whether has virulence in the template is detected at the upstream and downstream primer of the factor in the Aeromonas hydrophila that exists only in virulence, compared with prior art, since at the factor be to have the Aeromonas hydrophila strain of virulence peculiar, therefore can examine and determine the Aeromonas hydrophila strain that whether has virulence in the sample exactly, thereby improve the accuracy of disease early warning.Simultaneously, detect by said method one or method two and to contain hemolysin gene (hlyA) or serine protease gene (ahpA) in the template, it is necessarily pathogenic to show that then it contains, contain hemolysin gene (hlyA) and serine protease gene (ahpA) in the template and detect by method three, it is more highly pathogenic to show that then it contains.Three of method comes down to method one and method two same sample to be detected, and can determine that it is the virulent strain Aeromonas hydrophila when aimed strain has 2 kinds of virulence genes simultaneously; Can determine that it is the low virulent strain Aeromonas hydrophila when only having one of 2 kinds of virulence genes; 2 kinds of virulence genes can not determine that it is the non-virulent Hygrophilous monad when all detecting.

Method with FQ-PCR detection Aeromonas hydrophila provided by the invention, used following test kit: this test kit has in the test kit institute installed reagents:

1. 2x Premix EX Taq reaction solution: by Taq enzyme, buffer, dNTP Mixture, Mg ²⁺Mix;

2. upstream primer;

3. downstream primer;

4. TaqMan probe.

The Packing Unit of following reagent is the identical multiple of its experimental amount in the said test kit:

Experimental amount of 2x Premix EX Taq reaction solution is 10 μ l;

The concentration of upstream and downstream primer is 5 μ M, and an experimental amount is 0.4 μ l;

The concentration of TaqMan probe is 2.5 μ M, and an experimental amount is 0.4 μ l.

In the said test kit in the reagent of packaging, upstream and downstream primer and TaqMan probe have a group or two groups in first and second liang of groups, wherein the first group is: dna sequence dna is the upstream primer of 5 '-ACC GCA AGA TCA ACG ATA CCG AGT-3 ', dna sequence dna is the downstream primer of 5 '-ATC CAG CGA GAT CCG CAC TAT CTT-3 ', and dna sequence dna is the TaqMan probe of 5 '-(FAM)-AAC ATC TCG CTG GTT TAC CGG GTC AA-(TAMRA)-3 '; The second group is: dna sequence dna is the upstream primer of 5 '-ACC CAG GAC GGT GCG ATA TAA GAT-3 ', dna sequence dna is the downstream primer of 5 '-TCT CAC TTG CCT GAA CTGAAG CGA-3 ', and dna sequence dna is the TaqMan probe of 5 '-(FAM)-AAC ATC GTT AGC GTT GGC AAT CTC GG-(TAMRA)-3 '; The Packing Unit of 2x Premix EX Taq reaction solution is the twice of other reagent react amounts when having two groups.Be that 2x Premix EX Taq reaction solution can satisfy two groups of upstream and downstream primers and the needed amount of TaqMan probe reaction in the test kit.

Embodiment

The detection concrete steps of the inventive method are:

One, DNA extraction (template preparation) in the sample:

1. sample thief suspension supernatant 1ml joins in the aseptic eppendorf centrifuge tube of 1.5ml, is under the 15000r/min behind the centrifugal 2min at rotating speed, inhales and abandons supernatant, keeps precipitation;

2. add DNA extraction agent 50 μ l in the precipitation, will precipitate and DNA extraction agent mixing after be warmed up to 100 ℃ of heat treated 2min.Said DNA extraction agent is that 0.9% NaCl, percent weight in volume are that 0.01% SDS, volume percent are 0.1% beta-mercaptoethanol, all the other constitute for water by percent weight in volume;

3. continue to be centrifugal 2min under the 15000r/min at rotating speed, the gained supernatant is PCR need use template.

Two, PCR

1, amplification reaction solution is prepared:

1. from refrigerator, take out test kit;

Test kit has uses the seal-packed reagent of reagent bottle respectively, and packing list is:

5 μ M upstream primer first, 4 μ l, the dna sequence dna of upstream primer first are 5 '-ACC GCA AGA TCA ACG ATA CCG AGT-3 ';

5 μ M downstream primer first, 4 μ l, the dna sequence dna of downstream primer first are 5 '-ATC CAG CGA GAT CCG CAC TAT CTT-3 ';

2.5 μ M TaqMan probe first 4 μ l, the dna sequence dna of TaqMan probe first is 5 '-(FAM)-AAC ATC TCG CTG GTT TAC CGG GTC AA-(TAMRA)-3 ';

5 μ M upstream primer second, 4 μ l, the dna sequence dna of upstream primer second are 5 '-ACC CAG GAC GGT GCG ATA TAA GAT-3 ';

5 μ M downstream primer second, 4 μ l, the dna sequence dna of downstream primer second are 5 '-TCT CAC TTG CCT GAA CTG AAG CGA-3 ';

2.5 μ M TaqMan probe second 4 μ l, the dna sequence dna of TaqMan probe second is 5 '-(FAM)-AAC ATC GTT AGC GTT GGC AAT CTC GG-(TAMRA)-3 ';

2x Premix EX Taq reaction solution 200 μ l;

Aseptic double-distilled water 136 μ l;

DNA extraction agent 100 μ l also are packaged in the test kit, are used for the template preparation.

2. (include Taq enzyme, buffer, dNTP Mixture, Mg according to 2x Premix EX Taq reaction solution 10 μ l required in each PCR reaction tubes ²⁺Deng), 5 μ M upstream primers, 5 μ M downstream primers, 2.5 μ M TaqMan probes each 0.4 μ l, aseptic double-distilled water 6.8 μ l, by the required reaction tubes number of experiment, calculate required amount of reagent and add successively, the vortex mixing installs to the PCR reaction with in the glass capillary by every pipe 18 μ l branch.

2. application of sample:

1. install the PCR reaction of amplification reaction solution with adding 2 μ l templates in the glass capillary to each branch;

2. building PCR glass capillary lid, is centrifugal 30s under the 2000r/min at rotating speed then.

3, go up machine:

1. start, and carry out the instrument performance self check;

2. ready PCR reaction is placed on instrument sample aperture corresponding position with glass capillary;

3. on the PCR instrument, set following parameter:

Pre-sex change: 95 ℃ of circulations in 30 seconds;

Amplification: 63 ℃ of totally 40 circulations in 20 seconds are extended in 95 ℃ of sex change 5 seconds, annealing;

Collect fluorescent signal: 63 ℃, collection mode is made as " single-point is collected (SINGLE) ";

Instrument detecting passage: F1;

Rate temperature change: 20 ℃/second.

4. operation is determined in selected back, begins to carry out pcr amplification.

4, threshold setting and detected result are judged:

1. the threshold setting principle is for surpassing the vertex of negative control amplification line (random noise line).

2. contain Aeromonas hydrophila hlyA gene in some prompting sample of ct value＜35, the ct value is measured after prompting for uncertain sample between 35 ~ 40 need increase bacterium again once more.

3. not having the ct value shows then to point out and does not contain Aeromonas hydrophila hlyA gene in the sample or quantity seldom is lower than detectability.

During 4. as if the need detection by quantitative, the standard substance and the sample that add 4 ~ 5 different concns detect simultaneously, quantitative real time PCR Instrument can be depicted as the quantitative data that instrument behind the typical curve calculates positive sample automatically according to the ct value that each standard substance record, and on computer interface display density.

Negative and positive control can not show correctly that ct value judges that then detection fails.

Above-mentioned detection step is a single detection step of the present invention, i.e. said method one of the present invention and method two carry out with this detection step, and method three is carried out parallel test with this detection step respectively.

The inventive method is used for the preparation of Aeromonas hydrophila template DNA and was respectively 5 minutes and 17 minutes with the amplified reaction required time, receives result's report from sample and only needs about 30 minutes.And the real-time fluorescence quantitative PCR detection whole process that is used for the classics of Aeromonas hydrophila detection in the prior art still needs about 3 hours usually.

Sensitivity of the inventive method and batch interior repeatability are all very strong, aspect specific degree, the inventive method, except Aeromonas hydrophila, in 40 circulations to Vibrio parahaemolyticus (ATCC33847, ATCC17802), streptococcus aureus (ATCC25723, ATCC29213), escherichia coli (ATCC25922), Aeromonas caviae, Aeromonas sobria, the Maxwell vibrios, the O139 group cholera vibrio, the O1 group cholera vibrio, vibrio alginolyticus, Vibrio vulnificus, NAG vibrios (NAG), pseudomonas aeruginosa, paratyphoid bacillus A, Salmonella anatis, Shigella flexneri, the Song Shi shigella, enterorrhagia type escherichia coli O157 (933), the pure bacterium DNA extraction liquid (template) of Listeria monocytogenes does not all have the amplification phenomenon.

A kind of method with FQ-PCR detection Aeromonas hydrophila among the present invention is at the hlyA gene.There is 2x Premix EX Taq reaction solution (to include Taq enzyme, buffer, dNTP Mixture, Mg in the used kit ²⁺Deng) 100 μ l, 5 μ M dna sequence dnas are that upstream primer, the 5 μ M dna sequence dnas of 5 '-ACC GCA AGA TCA ACG ATA CCG AGT-3 ' are the downstream primer of 5 '-ATC CAG CGA GAT CCG CAC TAT CTT-3 ', each 4 μ l of TaqMan probe that 2.5 μ M dna sequence dnas are 5 '-(FAM)-AAC ATC TCG CTG GTT TAC CGG GTC AA-(TAMRA)-3 ', be sealed in respectively in the different reagent bottles.Working method is undertaken by above-mentioned concrete steps.

Present method rapid fluorescence quantitative PCR detection sensitivity: detect and be limited to 5.4 * 10 ¹Cfu/ml is 5.4 * 10 ³Cfu/ml-5.4 * 10 ⁸The scope internal standard curve of cfu/ml is good linear relationship.

Repeatability in its batch: the Aeromonas hydrophila dna sample of 4 parts of different concns, each concentration are divided into 5 parts to be tested, by the average with measured value

Calculate the standard deviation (s) and the variation coefficient (cv) of ct value and verify that batch interior repeatability is listed in the table below:

With regular-PCR as the reference method, 55 parts of field samples are carried out parallel detection comparison to be found, 5 parts of pure bacterium of Aeromonas hydrophila are strong positive, 5 parts of tap water are negative, it is in full accord that the positive rate of 3 parts of reservoir water, 6 parts of lake water, 6 parts of rivers, 20 portions of freshwater fishes is 84.0%, two kind of methods and results.15 parts of natural water-likes (3 parts of reservoir water, 6 parts of lake water, 6 parts of rivers) detected result is: the positive rate of real-time fluorescence PCR and regular-PCR is respectively 66.60% (10/15) and 60.0% (9/15), the water sample of regular-PCR test positive all is judged to the positive in fluorescent PCR, and regular-PCR detect negative water sample in fluorescent PCR, be judged to p+ 1 part of water sample be since in the water sample purpose bacterial content cause very little.

A kind of method with FQ-PCR detection Aeromonas hydrophila among the present invention is at the aphA gene.There is 2x Premix EX Taq reaction solution (to include Taq enzyme, buffer, dNTP Mixture, Mg in the used kit ²⁺Deng) 100 μ l, 5 μ M dna sequence dnas are the upstream primer of 5 '-ACC CAG GAC GGT GCG ATA TAA GAT-3 ', 5 μ M dna sequence dnas are the downstream primer of 5 '-TCT CAC TTG CCT GAA CTG AAG CGA-3 ', 2.5 μ M dna sequence dna is each 4 μ l of TaqMan probe of 5 '-(FAM)-AAC ATC GTT AGC GTT GGC AAT CTC GG-(TAMRA)-3 ', is sealed in respectively in the different reagent bottles.Working method is undertaken by above-mentioned concrete steps.

Present method rapid fluorescence quantitative PCR detection sensitivity: detect and be limited to 5.0 * 10 ¹Cfu/ml is 5.0 * 10 ³Cfu/ml-5.0 * 10 ⁷The scope internal standard curve of cfu/ml is good linear relationship.

Repeatability in batch: the Aeromonas hydrophila dna sample of 5 parts of different concns, each concentration are divided into 3 parts to be tested, by the average with measured value

Calculate the standard deviation (s) and the variation coefficient (cv) of ct value and verify that batch interior repeatability is listed in the table below:

As: in the dying Trionyx sinensis (Wiegmann) body of tool typical case's white abdominal shell disease symptom that the body weight that 1#, 2#, 6#, 9# (GenBank accession number GU563993, GU563992, GU563994, GU563995) all are located away to be provided by Zhejiang Province Aquatic Products Spreading Center, Yuyao City is 560.3g, be stored in Oceanography Institute Of Zhejiang's aquaculture laboratory safety locellus behind the purifying.Detect with above-mentioned test kit and by above-mentioned concrete steps respectively, all detect them and contain aphA gene and hlyA gene, except that the aphA gene copy amount of 9# bacterial strain and positive control near, all the other bacterial strains all are higher than positive control, negative control does not then detect.

Claims (2)

1. test kit that detects Aeromonas hydrophila with FQ-PCR is characterized in that having in the test kit institute installed reagents:

1. 2x Premix EX Taq reaction solution: by Taq enzyme, buffer, dNTP Mixture, Mg ²⁺Mix;

2. upstream primer;

3. downstream primer;

4. TaqMan probe;

Said upstream primer, downstream primer and TaqMan probe have a group or two groups in first and second liang of groups, wherein:

The dna sequence dna of first group upstream primer is 5 '-ACC GCA AGA TCA ACG ATA CCG AGT-3 ', the dna sequence dna of first group downstream primer is 5 '-ATC CAG CGA GAT CCG CAC TAT CTT-3 ', and the dna sequence dna of first group TaqMan probe is 5 '-(FAM)-AAC ATC TCG CTG GTT TAC CGG GTC AA-(TAMRA)-3 ';

The dna sequence dna of second group upstream primer is 5 '-ACC CAG GAC GGT GCG ATA TAA GAT-3 ', the dna sequence dna of downstream primer is 5 '-TCT CAC TTG CCT GAA CTG AAG CGA-3 ', and the dna sequence dna of TaqMan probe is 5 '-(FAM)-AAC ATC GTT AGC GTT GGC AAT CTC GG-(TAMRA)-3 '.

2. test kit as claimed in claim 1 is characterized in that the identical multiple of the Packing Unit of following reagent in the said test kit for its experimental amount:

Experimental amount of 2x Premix EX Taq reaction solution is 10 μ l;

The concentration of upstream and downstream primer is 5 μ M, and an experimental amount is 0.4 μ l;

The concentration of TaqMan probe is 2.5 μ M, and an experimental amount is 0.4 μ l;

The Packing Unit of 2x Premix EX Taq reaction solution is the twice of other reagent react amounts when having two groups.