CN1796571A - Method of multiplex fluorescence PCR - improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source - Google Patents
Method of multiplex fluorescence PCR - improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source Download PDFInfo
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Abstract
This invention relates to a method of detection of food-borne pathogenic bacteria using multiple fluorescent PCR and modified molecular beacons. Several pairs of primers and modified molecular beacon probes are designed according to the specific gene sequence of the pathogenic bacteria to be tested, which is amplified using fluorescent PCR and modified molecular beacon. The amplified product is crossbred with molecular beacon, and the fluorescent intensity of the reaction system is tested with the necessary circle times for intended threshold as a judgment for results, that is, negative for Ct of 0 or 40 and positive for Ct less than 38. This invention, which is advanced in high sensibility, significant specificity, simple operation and intuitionistic observation, is applicable in simultaneous detection of any two kinds, three kinds and four kinds of randomly combined bacteria and food-borne pathogenic bacteria as well as specimen in large amounts.
Description
Technical field
The present invention relates to the method for fluorescent PCR molecular beacons detection food-borne pathogens.
Background technology
The food origin disease sickness rate is higher, and for example vibrio cholerae causes cholera, and salmonella typhi and Shigellae cause typhoid fever and dysentery respectively.Cholera, typhoid fever, dysentery belong to the emphasis transmissible disease of China.The food poisoning that Vibrio parahaemolyticus, Salmonellas, streptococcus aureus, Bacillus cereus, Bacillus proteus etc. cause, its sickness rate accounts for higher ratio in China's food origin disease sickness rate.These food-borne pathogens still detect with the method for traditional bacterium separation and Culture and evaluation at present.This traditional detection method complex operation takes time and effort, and generally needs 5 day time, the longest time that also needs one month, and biochemical test and serological test instability; Detection sensitivity is low.
Along with the development of Protocols in Molecular Biology, people adopt round pcr to be applied to the diagnosis of bacterium.For example the Chinese patent publication number is the patent application of CN1526825, has disclosed a kind of specificity of utilizing Vibrio parahaemolyticus pR72H sequence, detects the method for Vibrio parahaemolyticus by modern molecular biology means such as DNA extraction, pcr amplification, electrophoresis observations.Yet this round pcr easily pollutes, and causes and detects failure, also needs electrophoresis.After nineteen ninety-five U.S. PE company proposes PCR in real time and detects principle, PCR in real time with fast, quantitatively, need not outstanding advantage such as rear electrophoresis, no crossed contamination and be widely adopted.
Molecular beacon is a kind of molecular probe with neck ring configuration that Tyagi in 1996 etc. put forward, be based on nucleic acid base pair principle and fluorescence resonance energy phenomenon the design, annealing stage at pcr amplification, molecular beacon combines with the target sequence of generation and sends fluorescence, in the extension stage, then break away from target sequence and do not disturb amplification, along with the increase of cycle index, also increase with the amount of template bonded molecular beacon, final fluorescence intensity is directly proportional with the template amount.
But currently used real-time PCR method is single fluorescence PCR method, can only detect a kind of pathogenic bacterium, and detection speed and sensitivity still can not meet the demands.Along with the variation and the antibiotic abuse of ecotope, many microbial clinical symptom of causing a disease more and more are not true to type, and usually need to detect simultaneously various bacteria and could determine cause of disease.This just has higher requirement to the quick diagnosis technology: not only fast but also can detect multiple pathogenic micro-organism simultaneously.Therefore be badly in need of the exploitation method of quick diagnosis various bacteria simultaneously.
Summary of the invention
In order to overcome shortcomings such as prior art complex operation, length consuming time, and can not detect the defective of multiple food-borne pathogens apace simultaneously and the method for a kind of multiple fluorescence PCR-improvement molecular beacons detection food-borne pathogens is provided.
For solving the problems of the technologies described above, technical scheme of the present invention is, the method for a kind of multiple fluorescence PCR-improvement molecular beacons detection food-borne pathogens, and it may further comprise the steps:
(1) many according to the special gene sequence design of various pathogenic bacterium to be measured to primer and a plurality of improvement molecular beacon probe;
(2) reaction system of preparation fluorescent PCR-improvement molecular beacon amplification gene sequence;
(3) with increase the simultaneously sequence of various pathogens specific gene to be measured of the various fluorescent PCRs-improvement molecular beacon of step (1);
(4) utilize the hybridization of molecular beacon and amplified production, the fluorescence intensity of detection reaction system, the standard that required cycle index Ct value is as a result of judged when reaching preset threshold, the Ct value is 0 or 40: feminine gender; Ct is less than 38: the positive.
The reaction system of fluorescent PCR-improvement molecular beacon amplification gene sequence is:
1 * PCR damping fluid
MgCl
2 0.5～5mmol
Each 0.05～1.5mmol of dNTP
Each is to primer 0.1～1.0 μ mol+0.1～1.0 μ mol
Each bar probe 0.1～1.0 μ mol
Taq enzyme 0.2～10U
Template 1～20 μ L
Cumulative volume 20～100 μ L
The reaction conditions of described fluorescent PCR-improvement molecular beacon amplifying specific gene order is 90～100 ℃ of pre-sex change 3～10 minutes, and 92～95 ℃ of sex change are 15～60 seconds in 40～50 circulations, 50～60 ℃ of annealing 15～60 seconds, and 70～72 ℃ were extended 15～120 seconds.
Described pathogenic bacterium are for being selected from vibrio cholerae, Vibrio parahaemolyticus, streptococcus aureus, singly increasing listeria spp, Salmonellas, Shigellae, and one or more the combination in the O157:H7 intestinal bacteria.
The special gene sequence of described pathogenic bacterium is respectively the heat stable nuclease gene NUC of vibrio cholerae enterotoxin genes ctxA sequence, the heat-resisting direct hemolysin gene of Vibrio parahaemolyticus TDH sequence, streptococcus aureus, singly increases the sequence of the hemolysin gene hly of listeria spp, the invasin gene ivnA sequence of Salmonellas, the coding aggressive plasmid antigen H gene ipaH sequence of Shigellae and the colibacillary hemolysin gene rfbE of O157:H7 sequence.
The primer and the probe of described improvement molecular beacon are respectively:
The a pair of primer of ctxA gene:
ctxA-F：5′-TCCGGAGCATAGAGCTTGGA-3′，
ctxA-R：5′-TCGATGATCTTGGAGCATTCC-3′
The probe of ctxA gene:
HEX-5′-
CCGTGGATTCATCATGCACCGCCACGG-3′Dabcyl，
The a pair of primer of TDH gene:
TDH-F：5′-AAACATCTGCTTTTGAGCTTCCA-3′，
TDH-R：5′-CTCGAACAACAAACAATATCTCATCAG-3′，
The probe of TDH gene:
FAM5′-CCGGGGTG
TCCCTTTTCCTGCCCCCGG-Dabcyl，
The a pair of primer of NUC gene:
NUC-F：5’-GGCAATACGCAAAGAGGTT-3’
NUC-L：5’-CTTCTTCTATTTACGCCGTTATC-3’
The probe of NUC gene:
ROX-5’-CGATGCA
GTCTAAGTAGCTCAGCAAATGCATCG-3’-DABCYL
The a pair of primer of hly gene:
hly-F：5’-TGCAAGTCCTAAGACGCCA-3’
hly-L：5’-CACTGCATCTCCGTGGTATACTAA-3’
The probe of hly gene:
FAM-5’-CGCG
CTTGTATATACTTATCGATTTCATCCGCGCG-3’-DABCYL
The a pair of primer of invA gene:
invA-F：5’-GCGGAATATC(I)ATGACGCAGCT-3’
invA-L：5’-CGCTACGTTTTGCTTCACGG-3’
The probe of invA gene:
5’-FAM-CCG
CCATTTGTATTGGTTGTTACGGCGG-DABCYL-3’
The a pair of primer of ipaH gene:
ipaH-F：5’-TGAAGGAAATGCGTTTCTATG-3’
ipaH-L：5’-AGGGAGAACCAGTCCGTAAA-3’
The probe of ipaH gene:
CY55’-CACG
GCCGAAGCTATGGTCAGAAGCCGTG-3’-DABCYL
The a pair of primer of rfbE gene:
rfbE-L：5’-AGGTGAAGGTGGAATGGTTGTC-3’
rfbE-R：5’-GCTTGTTCTAACTGGGCTAATC-3’
The probe of rfbE gene:
5’-FAMC
GGCCAAGGATTAGCTGTACATAGGCCG-DABCYL-3’
Wherein line part is for improveing the target sequence of molecular beacon probe.
Multiple fluorescence PCR-improvement molecular beacons detection food-borne pathogens is double, triple, quadruple or multiple fluorescence PCR-improvement molecular beacons detection food-borne pathogens.
The method of described multiple fluorescence PCR-improvement molecular beacons detection food-borne pathogens also further comprises to be handled and the template extraction step testing sample.
Described sample comprises food samples, ight soil, vomitus sample, what of ight soil and vomitus amount are wherein handled ight soil, vomitus sample and extract template is according to, suspend with 100-200 μ L physiological saline, boil, centrifugal 2 minutes of 10000rpm gets 5 μ L supernatant liquors and promptly can be used for real-time PCR reactions; The processing of food samples and the extraction of template are food samples is increased bacterium in the enrichment liquid at tested bacteria after, got 1.2mL enrichment liquid 10000rpm altogether centrifugal 5 minutes, after abandoning its supernatant liquor, add the 30uL tri-distilled water and boil, get 5 μ L supernatant liquors again and promptly can be used for real-time PCR reactions; Or add the lysate cracking, and through the phenol-chloroform extracting, and behind the ethanol sedimentation, add 20 μ L water dissolution, get 5 μ L solution and be used for real-time PCR reactions.
The invention has the beneficial effects as follows: the present invention has set up the method that multiple PCR in real time detects various bacteria simultaneously, this method on the basis of improvement molecular beacons technology:
(1) can be as required, double at random, triple, quadruple or Multiple Combination are tested various pathogens simultaneously; (2) highly sensitive, the 32-100cfu/mL bacterium can detect; (3) high specificity is with tens kinds of bacterium no cross reactions of control group; (4) simple to operate, the result observes simple and clear, but 96 parts of samples of one-time detection (containing the yin and yang attribute contrast); (5) detection speed is fast, detects stool and vomitus sample and only needs 2 hour detection time, detects the food sample and only needs the 1-2 days time (handle the early stage that comprises sample).
Description of drawings
Fig. 1 to Fig. 3 is fluorescent PCR of the present invention-improvement molecular beacons detection graphic representation.
Embodiment
The present invention is an example with the detection of food-borne pathogens, can make up at random, analyzes the detection method of multiple fluorescence PCR-improvement molecular beacon, thereby realizes detecting simultaneously rapidly and accurately various pathogens.
The improvement molecular beacon probe is in order to improve hybridization efficiency, to propose on original molecular beacon principle basis.The outstanding feature of improvement molecular beacon be arm portion with molecular beacon also as the target recognition sequence, and be not only the irrelevant interpolation sequence that is used to form hairpin structure.For the detection target sequence of same length, the improvement molecular beacon is shorter than molecule beacon, and because the arm sequence is no longer unsettled, thereby more be becoming tight close with combining of target sequence.The improvement molecular beacon is than the easier design of molecule beacon and have higher success rate, and simultaneously amplification condition required not strictly, detects in the time of therefore for a plurality of target sequence, and the advantage of improvement molecular beacon is more obvious.
The toxin gene of the food-borne pathogens of announcing according to GenBank or the conserved sequence of invasin gene, be invA, the Shigellae of TDH, the Salmonellas of ctxA, the Vibrio parahaemolyticus of vibrio cholerae ipaH, streptococcus aureus NUC and singly increase the hly gene of listeria spp, the colibacillary hemolytic plain gene of O157:H7 rfbE, design primer and improvement molecular beacon probe respectively, with different fluorescent agent label probes, compare with tens kinds of bacteriums simultaneously, set up improvement molecular beacon-fluorescent PCR and detect the reaction system of food-borne pathogens.And this detection architecture is applied to the quick diagnosis of common bacterial food poisoning.The method that detects food-borne pathogens with multiple fluorescence PCR mainly may further comprise the steps:
(1) toxin gene of the food-borne pathogens of announcing according to GenBank or the conserved sequence of invasin gene design primer and probe respectively;
(2) testing sample is handled and template extraction;
(3) fluorescent PCR amplification;
(4) detect fluorescent signal, the standard that required cycle index Ct value is as a result of judged when reaching preset threshold.
The heat stable nuclease gene NUC of the vibrio cholerae enterotoxin genes ctxA sequence of announcing according to GenBank in the step (1), the heat-resisting direct hemolysin gene of Vibrio parahaemolyticus TDH sequence, streptococcus aureus, singly increase the sequence of the hemolysin gene hly of listeria spp, the invasin gene ivnA sequence of Salmonellas, the coding aggressive plasmid antigen H gene ipaH sequence of Shigellae, and the colibacillary hemolytic gene of O157:H7 rfbE sequence, the primer and the probe of improvement molecular beacon of the present invention are respectively:
The a pair of primer of ctxA gene:
ctxA-F：5′-TCCGGAGCATAGAGCTTGGA-3′，
ctxA-R：5′-TCGATGATCTTGGAGCATTCC-3′
The probe of ctxA gene:
HEX-5′-
CCGTGGATTCATCATGCACCGCCACGG-3′Dabcyl，
The a pair of primer of TDH gene:
TDH-F：5′-AAACATCTGCTTTTGAGCTTCCA-3′，
TDH-R：5′-CTCGAACAACAAACAATATCTCATCAG-3′，
The probe of TDH gene:
FAM5′-CCGGGG
TGTCCCTTTTCCTGCCCCCGG-Dabcyl，
The a pair of primer of NUC gene:
NUC-F：5’-GGCAATACGCAAAGAGGTT-3’
NUC-L：5’-CTTCTTCTATTTACGCCGTTATC-3’
The probe of NUC gene:
ROX-5’-CGATGCA
GTCTAAGTAGCTCAGCAAATGCATCG-3’-DABCYL
The a pair of primer of hly gene:
hly-F：5’-TGCAAGTCCTAAGACGCCA-3’
hly-L：5’-CACTGCATCTCCGTGGTATACTAA-3’
The probe of hly gene:
FAM-5’-CGCG
CTTGTATATACTTATCGATTTCATCCGCGCG-3’-DABCYL
The a pair of primer of invA gene:
invA-F：5’-GCGGAATATC(I)ATGACGCAGCT-3’
invA-L：5’-CGCTACGTTTTGCTTCACGG-3’
The probe of invA gene:
5’-FAM-CCG
CCATTTGTATTGGTTGTTACGGCGG-DABCYL-3’
The a pair of primer of ipaH gene:
ipaH-F：5’-TGAAGGAAATGCGTTTCTATG-3’
ipaH-L：5’-AGGGAGAACCAGTCCGTAAA-3’
The probe of ipaH gene:
CY55’-CACG
GCCGAAGCTATGGTCAGAAGCCGTG-3’-DABCYL
The a pair of primer of rfbE gene:
rfbE-L：5’-AGGTGAAGGTGGAATGGTTGTC-3’
rfbE-R：5’-GCTTGTTCTAACTGGGCTAATC-3’
The probe of rfbE gene:
5’-FAMC
GGCCAAGGATTAGCTGTACATAGGCCG-DABCYL-3’。
Wherein line part is for improveing the target sequence of molecular beacon probe.
Step (2) sample preparation and template extraction, the sample scope of application comprises samples such as food samples, ight soil, vomitus.For ight soil, vomitus sample, according to ight soil and vomitus amount what, suspend with 100-200 μ L physiological saline, boil, centrifugal 2 minutes of 10000rpm gets 5 μ L supernatant liquors and promptly can be used for real-time PCR reactions.The processing of food samples and the extraction of template are food samples is increased bacterium in the enrichment liquid at tested bacteria after, get the 1.2mL enrichment liquid altogether centrifugal 5 minutes in 10000rpm, after abandoning its supernatant liquor, add the 30uL tri-distilled water and boil, get 5 μ L supernatant liquors again and promptly can be used for real-time PCR reactions, perhaps abandon and add the lysate cracking after its supernatant liquor, through the phenol-chloroform extracting, and behind the ethanol sedimentation, add 20 μ L water dissolution, get 5 μ L solution and be used for real-time PCR reactions.Therefore during with multiple fluorescence PCR-improvement molecular beacons detection food samples, will form a plurality of templates.
The fluorescent PCR amplification reaction system of step (3) is:
1 * PCR damping fluid
MgCl
2 0.5～5mmol
Each 0.05～1.5mmol of dNTP
Each is to primer 0.1～1.0 μ mol+0.1～1.0 μ mol
Each bar probe 0.1～1.0 μ mol
Taq enzyme 0.2～10U
Template 1～20 μ L
Cumulative volume 20～100 μ L
Above reagent component: 1 * PCR damping fluid, MgCl
2, Taq enzyme, dNTP are all available from the precious biotech firm of DaLian, China.
Reaction conditions is: 90～100 ℃ of pre-sex change 3～10 minutes, and 92～95 ℃ of sex change are 15～60 seconds in 40～50 circulations, 50～60 ℃ of annealing 15～60 seconds, 72 ℃ were extended 15～120 seconds down.
Step (4) detects the Ct value of fluorescent signal, be to adopt iCycler PCR in real time amplification instrument (Bio-Rad company) or MX4000 fluorescent PCR amplification instrument or Rotor fluorescent PCR amplification instrument annealing stage to detect fluorescence, once can detect 96 duplicate samples (comprising the negative and positive contrast).Ct value judged result according to the demonstration of fluorescent PCR amplification instrument: the Ct value is that 0 or 40 expressions are negative; Ct is positive less than 38 expressions; Ct is between 38-40 the time, and sample needs to detect again.Detect stool and vomitus sample and only need 2 hour detection time, detect the food sample and only need the 1-2 days time (handle the early stage that comprises sample).
Example one
Be the method that example is analyzed multiple fluorescence PCR-improvement molecular beacons detection food-borne pathogens with double fluorescent PCR-improvement molecular beacons detection Salmonellas and Shigellae in this example.The sequence and the comparative analysis of invasin gene (ivnA) of the Salmonellas of announcing according to GenBank and the coding aggressive plasmid antigen H gene (ipaH) of Shigellae, She Ji a pair of primer and probe is described with preamble respectively.
Bacterial strain is used in test: 14 kinds of bacteriums are totally 132 strain bacterial strains.Comprise 1 strain enterotoxigenic E (enterotoxigenic E.coli, ETEC), 1 strain enteroinvasive E (enteroinvasive E.coli, EIEC), 1 strain O157:H7 intestinal bacteria (O157:H7), 1 strain pathogenic colon bacillus (enteropathogenic E.coli, EPEC), 1 strain citrobacter (Citrobacter), 1 strain colon bacillus E.coli, 1 strain Vibrio parahaemolyticus (V.parahaemolyticus), 1 strain cholera (V.cholera) vibrios, 1 strain streptococcus aureus (S.aureus), 1 strain singly increases listeria spp (Listeria monocytogenes), 1 strain Bacillus cereus (B.cereus), 1 strain Bacillus proteus (Proteus), 70 strain Salmonellass (Salmonella) (various serotype), 50 strain Shigellaes (Shigella) (various serotype).All bacterial strains all separate according to " food microbiology " national inspecting standard of People's Republic of China's promulgation and identify.
It is further comprising the steps of to utilize above-mentioned fluorescence PCR method to detect the method for Salmonellas and Shigellae simultaneously:
(1) sample preparation and template extraction: the sample scope of application comprises samples such as food samples, ight soil, vomitus; Processing to ight soil, vomitus sample is identical with extraction and preceding method; For food samples, when wherein detecting Salmonellas, 25g food is placed on increases bacterium 10 hours among the 225mLSC; For Shigellae, 25g food be placed on increase bacterium 10 hours among the 225mLGN, increase bacterium after, get the 1mL enrichment liquid, centrifugal 5 minutes of 10000rpm, abandon its supernatant liquor after, add the 30uL tri-distilled water and boil, get 5 μ L supernatant liquors again and promptly can be used for real-time PCR reactions.
(2) fluorescent PCR amplification, preparation multiple fluorescence PCR reaction system:
1 * PCR damping fluid
MgCl
2 1.5mmol
Each 0.05mmol of dNTP
The primer 0.2 μ mol+0.2 μ mol of invA gene
The primer 0.2 μ mol+0.2 μ mol of ipaH gene
The probe 0.2 μ mol of invA gene
The probe 0.2 μ mol of ipaH gene
Taq enzyme 1U
Template 10 μ L
Cumulative volume 25 μ L
Real-time PCR reactions: 95 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change are 30 seconds in 45 circulations, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 60 seconds.
(3) detect: adopt iCycler PCR in real time amplification instrument (Bio-Rad company) annealing stage to detect fluorescence, once can detect 96 duplicate samples (comprising the negative and positive contrast).The result judges: the Ct value judged result that shows according to fluorescent PCR amplification instrument: the Ct value is 0 or 40: feminine gender; Ct is less than 38: the positive; Ct is between 38-40: sample needs to detect again.
Aforementioned 14 kinds of bacteriums totally 132 strain bacterial strains detect through improvement molecular beacon system of the present invention, the Salmonellas that only contains the ivnA gene has fluorescent signal with the Shigellae that contains the ipaH gene, other bacteriums do not have fluorescent signal, have proved that further the specificity of fluorescent PCR sees Table 1.
The dual PCR in real time specificity analyses of table 1 Salmonellas and Shigellae
| Bacterium | Amount detection | The quantity of invA PCR in real time tests positive | The quantity of ipaH PCR in real time tests positive |
| Salmonellas | 70 | 70 | 0 |
| Shigellae | 50 | 0 | 50 |
| Enterotoxigenic E | 1 | 0 | 0 |
| Pathogenic colon bacillus | 1 | 0 | 0 |
| Enteroinvasive E | 1 | 0 | 0 |
| Colon bacillus | 1 | 0 | 0 |
| Citrobacter | 1 | 0 | 0 |
| Cholera | 1 | 0 | 0 |
| Vibrio parahaemolyticus | 1 | 0 | 0 |
| The O157:H7 intestinal bacteria | 1 | 0 | 0 |
| Bacillus cereus | 1 | 0 | 0 |
| Bacillus proteus | 1 | 0 | 0 |
| Singly increase listeria spp | 1 | 0 | 0 |
| Streptococcus aureus | 1 | 0 | 0 |
By table 1 as seen, fluorescent PCR of the present invention-improvement molecular beacon high specificity is with control group ten various bacteria no cross reactions.
With aforementioned 14 kinds of bacteriums totally 132 strain bacterial strains experimentize, detect the blank system that does not contain Salmonellas or Shigellae respectively, contain the system of Salmonellas and Shigellae, the system that contains the system of Salmonellas and contain Shigellae with single or multiple fluorescence PCR method of the present invention by the preceding method preparation.Adopt iCycler PCR in real time amplification instrument to detect annealing stage fluorescence, fluorescent signal curve such as Fig. 1.Wherein, Fig. 1 (a) is the fluorescent signal curve that the fluorescent PCR amplification instrument of blank system is measured, Fig. 1 (b) detects the fluorescent signal curve that contains Salmonellas and Shigellae for multiple fluorescence PCR, Fig. 1 (c) detects the fluorescent signal curve that contains Salmonellas for fluorescent PCR, and Fig. 1 (d) detects the fluorescent signal curve that contains Shigellae for fluorescent PCR.By Fig. 1 comparative analysis, multiple fluorescence PCR detects multiple bacterium, and no cross reaction does not disturb and restraining effect each other, and fluorescent signal does not weaken, high specificity.
Sensitivity analysis: select 1 strain Salmonellas and 1 strain Shigellae representative strains respectively as sensitivity analysis.Salmonellas bacterium liquid sensitivity analysis method: the Salmonellas bacterial strain carries out 10 times of dilutions after using SC enrichment liquid overnight incubation, does 10 extent of dilution altogether, gets 10 dilution 1mL bacterium liquid then successively and carries out real-time PCR reactions.Simultaneously corresponding " food microorganisms inspecting standard " by People's Republic of China's promulgation done total bacterial count.The sensitivity analysis of Shigellae bacterium liquid: after the Shigellae bacterial strain was used meat soup enrichment liquid overnight incubation, all the other steps were by the operation of " sensitivity analysis of Salmonellas bacterium liquid " method.The result shows that fluorescence PCR method of the present invention is highly sensitive, and the 32-100cfu/mL bacterium can detect.
Replica test: respectively 1 strain Salmonellas and the repetition of 1 strain Shigellae are carried out the fluorescent PCR amplification for 10 times, 10 times the Ct value differs less than 0.1 circulation.
Gather 350 duplicate samples altogether, comprise food samples and food poisoning sample.When detecting with fluorescent PCR, 350 duplicate samples adopt traditional bacterial culture to verify.As stated above sample is handled and the extraction of template after detect, traditional detection method and fluorescence PCR detecting method relatively see Table 2.
The comparison of table 2 traditional detection method and fluorescence PCR detecting method
| Test item | The sample classification | Sample size | Fluorescent PCR | Traditional method | ||
| Number positive | Detection time | Number positive | Detection time | |||
| Salmonellas | Stool | 50 | 30 | 2 hours | 30 | 4 days |
| Food | 200 | 65 | 1 day | 32 | 4 days | |
| Shigellae | Stool | 50 | 1 | 2 hours | 1 | 4 days |
| Food | 100 | 0 | 1 day | 0 | 4 days | |
The multiple fluorescence PCR method detects stool and the vomitus sample only needs 2 hour detection time, detects the food sample and only needs the 1 day time (handle the early stage that comprises sample), can detect various bacteria simultaneously.Therefore the multiple fluorescence PCR method is time saving and energy saving, and the accuracy height can satisfy the quick diagnosis of disease.And fluorescence PCR method and traditional detection method result's coincidence rate is 100%, and fluorescent PCR is highly sensitive in traditional detection method, and sample contains the 100cfu/mL bacterium and can detect.
Example two
Improveing molecular beacons detection Shigellae, O157:H7 intestinal bacteria, singly increase listeria spp with triple fluorescent PCR-in this example is example.Announce the colibacillary hemolysin gene rfbE of sequence, the O157:H7 sequence of the coding aggressive plasmid antigen H gene (ipaH) of Shigellae, singly increase the sequence of the hemolysin gene (hly) of listeria spp according to GenBank, She Ji a pair of primer and probe and amplified fragments thereof are described with preamble respectively.
Bacterial strain is used in test: O157:H7 intestinal bacteria (O157:H7), singly increase listeria spp (Listeria monocytogenes), Shigellae (Shigella) (various serotype).All bacterial strains all separate according to " food microbiology " national inspecting standard of People's Republic of China's promulgation and identify.
Utilize above-mentioned fluorescence PCR method to detect Shigellae, O157:H7 intestinal bacteria simultaneously, singly to increase the method for listeria spp further comprising the steps of:
(1) sample preparation and template extraction: the sample scope of application comprises samples such as food samples, ight soil, vomitus; To the processing of ight soil, vomitus sample and extracting method with example one; Food samples: for Shigellae, 25g food is placed on increases bacterium 10 hours among the 225mLGN, for the O157:H7 intestinal bacteria, 25g food is placed in the 225mL enteron aisle enrichment liquid increased bacterium 10 hours, for singly increasing listeria spp: 25g food is placed on and increased bacterium among the 225mLLB1 24 hours, after increasing bacterium, respectively get the 0.4mL enrichment liquid, be total to 1.2mL, centrifugal 5 minutes of 10000rpm, abandon its supernatant liquor, add the lysate cracking, through the phenol-chloroform extracting, and behind the ethanol sedimentation, add 20 μ L water dissolution, get 5 μ L solution and be used for real-time PCR reactions.
(2) reaction system of triple fluorescent pcr amplification:
1 * PCR damping fluid
MgCl
2 1.5mmol
Each 0.05mmol of dNTP
The primer 0.2 μ mol+0.2 μ mol of ipaH gene
The primer 0.2 μ mol+0.2 μ mol of rfbE gene
The primer 0.2 μ mol+0.2 μ mol of hly gene
The probe 0.2 μ mol of ipaH gene
The probe 0.2 μ mol of rfbE gene
The probe 0.2 μ mol of hly gene
Taq enzyme 1U
Template 10 μ L
Cumulative volume 25 μ L
Real-time PCR reactions: 95 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change are 30 seconds in 45 circulations, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 60 seconds.
(3) detect: adopt Rotor fluorescent PCR amplification instrument (Roche company) annealing stage to detect fluorescence, once can detect 96 duplicate samples (comprising the negative and positive contrast).The result judges: the Ct value judged result that shows according to fluorescent PCR amplification instrument: the Ct value is 0 or 40: feminine gender; Ct is less than 38: the positive; Ct is between 38-40: sample needs to detect again.
Detect with triple fluorescent PCR method of the present invention and to contain Shigellae, O157:H7 intestinal bacteria, singly increase the system of listeria spp, adopt iCycler PCR in real time amplification instrument to detect annealing stage fluorescence, record three fluorescent signal curves such as Fig. 2 simultaneously.Wherein, Fig. 2 (a) is the fluorescent signal curve that the fluorescent PCR amplification instrument of Shigellae system is measured, and the bar numerical table of curve shows sample number.Fig. 2 (b) is the colibacillary fluorescent signal curve of O157:H7, and Fig. 2 (c) contains the fluorescent signal curve that singly increases listeria spp for fluorescent PCR detects.By Fig. 2 comparative analysis, triple fluorescent PCR detects various bacteria, no cross reaction, high specificity.
The multiple fluorescence PCR method detects stool and the vomitus sample only needs 2 hour detection time, detects the food sample and only needs the 1-2 days time (handle the early stage that comprises sample), can detect various bacteria simultaneously.Therefore the multiple fluorescence PCR method is time saving and energy saving, and the accuracy height can satisfy the quick diagnosis of disease.And fluorescence PCR method and traditional detection method result's coincidence rate is 100%, and fluorescent PCR is highly sensitive in traditional detection method, and sample contains the 100cfu/mL bacterium and can detect.
Example three
This example explanation quadruple fluorescent PCR-improvement molecular beacon detects simultaneously and contains Shigellae, vibrio cholerae, streptococcus aureus, the colibacillary method of O157:H7.Announce sequence, the sequence of vibrio cholerae enterotoxin genes ctxA, the heat stable nuclease gene NUC sequence of streptococcus aureus, the colibacillary hemolytic plain gene of the O157:H7 rfbE sequence of the coding aggressive plasmid antigen H gene (ipaH) of Shigellae according to GenBank, She Ji a pair of primer and probe and amplified fragments are described with preamble respectively.
Test bacterial strain: Shigellae, vibrio cholerae, streptococcus aureus, O157:H7 intestinal bacteria.All bacterial strains all separate according to " food microbiology " national inspecting standard of People's Republic of China's promulgation and identify.
It is further comprising the steps of to utilize above-mentioned fluorescence PCR method to detect Shigellae, vibrio cholerae, streptococcus aureus, the colibacillary method of O157:H7 simultaneously:
(1) sample preparation and template extraction: the sample scope of application comprises samples such as food samples, ight soil, vomitus; To the processing of ight soil, vomitus sample and extracting method with example one and example two; For food samples: for Shigellae, 25g food is placed on increases bacterium 10 hours among the 225mLGN, for the O157:H7 intestinal bacteria, 25g food is placed in the 225mL enteron aisle enrichment liquid increased bacterium 10 hours, for streptococcus aureus: 25g food is placed on and increases bacterium 10 hours among the 225mL 7.5%NaCl, for vibrio cholerae, 25g food is respectively got the 0.3mL enrichment liquid after being placed on and increasing bacterium in the 225mL basic peptone water, be total to 1.2mL, centrifugal 5 minutes of 10000rpm abandons its supernatant liquor, adds the lysate cracking, through the phenol-chloroform extracting, and behind the ethanol sedimentation, add 20 μ L water dissolution, get 5 μ L solution and be used for real-time PCR reactions.
(2) quadruple fluorescent PCR amplification, preparation quadruple fluorescent PCR reaction system:
1 * PCR damping fluid
MgCl2 1.5mmol
Each 0.05mmol of dNTP
The primer 0.2 μ mol+0.2 μ mol of ipaH gene
The primer 0.2 μ mol+0.2 μ mol of ctxA gene
The primer 0.2 μ mol+0.2 μ mol of NUC gene
The primer 0.2 μ mol of rfbE gene
The probe 0.2 μ mol of ipaH gene
The probe 0.2 μ mol of ctxA gene
The probe 0.2 μ mol of NUC gene
The probe 0.2 μ mol of rfbE gene
Taq enzyme 1U
Template 10 μ L
Cumulative volume 25 μ L
The condition of real-time PCR reactions is with example one and example two.
(3) detect: adopt Rotor fluorescent PCR amplification instrument (Roche company) annealing stage to detect fluorescence, once can detect 96 duplicate samples (comprising the negative and positive contrast).The result judges: the Ct value judged result that shows according to fluorescent PCR amplification instrument: the Ct value is 0 or 40: feminine gender; Ct is less than 38: the positive; Ct is between 38-40: sample needs to detect again.
Contain Shigellae, vibrio cholerae, streptococcus aureus, the colibacillary system of O157:H7 with quadruple fluorescence PCR method detection of the present invention.Adopt iCycler PCR in real time amplification instrument to detect annealing stage fluorescence, fluorescent signal curve such as Fig. 3.Wherein, Fig. 3 (a) is the fluorescent signal curve that the fluorescent PCR amplification instrument of Shigellae system is measured, and the bar numerical table of curve shows sample number.Fig. 3 (b) is the fluorescent signal curve of vibrio cholerae, and Fig. 3 (c) is the fluorescent signal curve of streptococcus aureus, and Fig. 3 (d) is the colibacillary fluorescent signal curve of O157:H7.By Fig. 3 comparative analysis, the quadruple fluorescent PCR detects multiple bacterium, no cross reaction, high specificity.
Various other food-borne pathogens, as Salmonellas, Shigellae, streptococcus aureus, Bacillus cereus, singly increase listeria spp, Bacillus proteus, Hemolytic streptococcus, Vibrio parahaemolyticus and vibrio cholerae etc., through dual, triple, quadruples etc. make up at random, detect with above-mentioned multiple fluorescence PCR-improvement molecular beacons detection method, adopt iCycler PCR in real time amplification instrument (Bio-Rad company) annealing stage to detect fluorescence, various food-borne pathogens detect high specificity all appears and to the similar curve of aforementioned fluorescent signal curve, and detection sensitivity height.
The detection architecture of multiple fluorescence PCR-improvement molecular beacons detection food-borne pathogens can be developed into a sequence quick detection kit, satisfy the routine check of Disease Prevention and Control Institutions, inspection and quarantine department and technical supervision department and the quick reply of Public Health Emergencies, have important social benefit and economic benefit.
Claims (9)
1, the method for a kind of multiple fluorescence PCR-improvement molecular beacons detection food-borne pathogens, it may further comprise the steps:
(1) special gene sequence according to various pathogenic bacterium to be measured designs many to primer and a plurality of improvement molecular beacon probe respectively;
(2) reaction system of preparation fluorescent PCR-improvement molecular beacon amplification gene sequence;
(3) with increase the simultaneously sequence of various pathogens specific gene to be measured of the various fluorescent PCRs-improvement molecular beacon of step (1);
(4) utilize the hybridization of molecular beacon and amplified production, the fluorescence intensity of detection reaction system, the standard that required cycle index Ct value is as a result of judged when reaching preset threshold, the Ct value is 0 or 40: feminine gender; Ct is less than 38: the positive.
2, the method for multiple fluorescence PCR as claimed in claim 1-improvement molecular beacons detection food-borne pathogens is characterized in that: the reaction system of described fluorescent PCR-improvement molecular beacon amplification gene sequence is:
1 * PCR damping fluid
MgCl
2 0.5～5mmol
Each 0.05～1.5mmol of dNTP
Each is to primer 0.1～1.0 μ mol+0.1～1.0 μ mol
Each bar probe 0.1～1.0 μ mol
Taq enzyme 0.2～10U
Template 1～20 μ L
Cumulative volume 20～100 μ L
3, the method for multiple fluorescence PCR as claimed in claim 1-improvement molecular beacons detection food-borne pathogens, it is characterized in that: the reaction conditions of described fluorescent PCR-improvement molecular beacon amplifying specific gene order is 90～100 ℃ of pre-sex change 3～10 minutes, 92～95 ℃ of sex change are 15～60 seconds in 40～50 circulations, annealed 15～60 seconds for 50～60 ℃, 70～72 ℃ were extended 15～120 seconds.
4, as the method for each described multiple fluorescence PCR-improvement molecular beacons detection food-borne pathogens in the claim 1 to 3, it is characterized in that: described pathogenic bacterium are for being selected from vibrio cholerae, Vibrio parahaemolyticus, streptococcus aureus, singly increasing listeria spp, Salmonellas, Shigellae, and one or more the combination in the O157:H7 intestinal bacteria.
5, the method of multiple fluorescence PCR as claimed in claim 4-improvement molecular beacons detection food-borne pathogens, it is characterized in that: the special gene sequence of described pathogenic bacterium is respectively vibrio cholerae enterotoxin genes ctxA sequence, the heat-resisting direct hemolysin gene of Vibrio parahaemolyticus TDH sequence, the heat stable nuclease gene NUC of streptococcus aureus, singly increase the sequence of the hemolysin gene hly of listeria spp, the invasin gene ivnA sequence of Salmonellas, the coding aggressive plasmid antigen H gene ipaH sequence of Shigellae, and the colibacillary hemolysin gene rfbE of O157:H7 sequence.
6, the method for multiple fluorescence PCR as claimed in claim 5-improvement molecular beacons detection food-borne pathogens, it is characterized in that: the primer and the probe of described improvement molecular beacon are respectively:
The a pair of primer of ctxA gene:
ctxA-F：5′-TCCGGAGCATAGAGCTTGGA-3′，
ctxA-R：5′-TCGATGATCTTGGAGCATTCC-3′
The probe of ctxA gene:
HEX-5′-
CCGTGGATTCATCATGCACCGCCACGG-3′Dabcyl，
The a pair of primer of TDH gene:
TDH-F：5′-AAACATCTGCTTTTGAGCTTCCA-3′，
TDH-R：5′-CTCGAACAACAAACAATATCTCATCAG-3′，
The probe of TDH gene:
FAM5′-CCGGGG
TGTCCCTTTTCCTGCCCCCGG-Dabcyl，
The a pair of primer of NUC gene:
NUC-F：5’-GGCAATACGCAAAGAGGTT-3’
NUC-L：5’-CTTCTTCTATTTACGCCGTTATC-3’
The probe of NUC gene:
ROX-5’-CGATGCA
GTCTAAGTAGCTCAGCAAATGCATCG-3’-DABCYL
The a pair of primer of hly gene:
hly-F：5’-TGCAAGTCCTAAGACGCCA-3’
hly-L：5’-CACTGCATCTCCGTGGTATACTAA-3’
The probe of hly gene:
FAM-5’-CGCG
CTTGTATATACTTATCGATTTCATCCGCGCG-3’-DABCYL
The a pair of primer of invA gene:
invA-F：5’-GCGGAATATC(I)ATGACGCAGCT-3’
invA-L：5’-CGCTACGTTTTGCTTCACGG-3’
The probe of invA gene:
5’-FAM-CCG
CCATTTGTATTGGTTGTTACGGCGG-DABCYL-3’
The a pair of primer of ipaH gene:
ipaH-F：5’-TGAAGGAAATGCGTTTCTATG-3’
ipaH-L：5’-AGGGAGAACCAGTCCGTAAA-3’
The probe of ipaH gene:
CY55’-CACG
GCCGAAGCTATGGTCAGAAGCCGTG-3’-DABCYL
The a pair of primer of rfbE gene:
rfbE-L：5’-AGGTGAAGGTGGAATGGTTGTC-3’
rfbE-R：5’-GCTTGTTCTAACTGGGCTAATC-3’
The probe of rfbE gene:
5 '-FAMC
GGCCAAGGATTAGCTGTACATAGGCCG-DABCYL-3 ', wherein line part is for improveing the target sequence of molecular beacon probe.
7, the method for multiple fluorescence PCR as claimed in claim 1-improvement molecular beacons detection food-borne pathogens is characterized in that: described multiple fluorescence PCR-improvement molecular beacons detection food-borne pathogens is dual, triple or quadruple fluorescent PCR-improvement molecular beacons detection food-borne pathogens.
8, the method for multiple fluorescence PCR as claimed in claim 1-improvement molecular beacons detection food-borne pathogens is characterized in that: also further comprise testing sample is handled and the template extraction step.
9, the method for multiple fluorescence PCR as claimed in claim 8-improvement molecular beacons detection food-borne pathogens, it is characterized in that: described sample comprises food samples, ight soil or vomitus sample, what of ight soil and vomitus amount are wherein handled ight soil, vomitus sample and extract template is according to, suspend with 100-200 μ L physiological saline, boil, centrifugal 2 minutes of 10000rpm gets 5 μ L supernatant liquors and promptly can be used for real-time PCR reactions; The processing of food samples and the extraction of template are food samples is increased bacterium in the enrichment liquid at tested bacteria after, got 1.2mL enrichment liquid 10000rpm altogether centrifugal 5 minutes, after abandoning its supernatant liquor, add the 30uL tri-distilled water and boil, get 5 μ L supernatant liquors again and promptly can be used for real-time PCR reactions, or add the lysate cracking after abandoning its supernatant liquor, through the phenol-chloroform extracting, and behind the ethanol sedimentation, add 20 μ L water dissolution, get 5 μ L solution and be used for real-time PCR reactions.
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