CN101948923B - Method and kit for detecting clostridium tetani by using loop-mediated isothermal amplification method - Google Patents
Method and kit for detecting clostridium tetani by using loop-mediated isothermal amplification method Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, and particularly discloses a method and a kit thereof for detecting clostridium tetani by using a loop-mediated isothermal amplification method. The method comprises the following steps of: amplifying clostridium tetani spasm toxin genes in a sample DNA by using loop-mediated isothermal amplification technology and using an upstream inner primer, a downstream inner primer, an upstream outer primer and a downstream outer primer, wherein the nucleotide sequences of the primers are expressed as SEQ ID No: 1-4; and detecting the amplification results. The kit comprises the upstream inner primer, the downstream inner primer, the upstream outer primer, the downstream outer primer, DNA quick extraction liquid and isothermal amplification liquid. The method and the kit thereof for detecting the clostridium tetani by using the loop-mediated isothermal amplification method have the advantages of high clostridium tetani specificity, high sensitivity and simple and quick detection process.
Description
Technical field
The present invention relates to biology field, relate to a kind of method in particular, also relate to a kind of test kit that detects tetanus bacillus with the loop-mediated isothermal amplification method with loop-mediated isothermal amplification method detection tetanus bacillus.
Background technology
Tetanus is to infect the transmissible disease that tetanus bacillus causes, is to invade people's human body wound and breeding and produce the caused a kind of acute specific infection of extracellular toxin in wound by tetanus bacillus. tic is main with paroxysmal with patient's whole body or local muscle fixed spasm clinically.
Tetanus bacillus is a kind of obligate anaerobic property clostridium, and Gram-positive belongs to conditioned pathogen, germ only under the condition of anaerobic or wound is dark and situation that infect with the aerobic bacterium under be prone to growth and breeding.Tetanus bacillus is present in people and animals' enteron aisle at ordinary times, excretes with ight soil, and in nature, to be common in the soil, this bacterium has very strong drag to environment especially with brood cell's distributions, can be high temperature resistant.Tetanus bacillus and toxin thereof can not be invaded normal skin and mucous membrane, so after tetanus all occurs in wound.All open injuries all have tetanic possibility take place, like wound, industrial injury, burn, frostbite etc. after war wound, the earthquake.After possibly occurring in various wounds, tetanus also possibly betide puerpera and the newborn infant who gives a birth under the unclean condition.
Tetanus bacillus can produce a kind of extracellular toxin after acting on human body; Make human body show the very violent muscle of mastication and the tic of trunk muscles, the patient usually can mandibular joint tightly stings, health is in spite of oneself toward layback, external environment sometimes; Minimal irritation like sound, light, vibrations; Just can cause the violent tic of patient and a large amount of perspirations, serious patient also can cause the paralysis of breathing, causes death.
Therefore, detect tetanus bacillus early help to prevent tetanic generation.At present, the evaluation of tetanus bacillus mainly is that secretory product direct smear, foranalysis of nucleic acids, methods,anaerobic and automatic bacterial culture identification appearance are identified.Method of direct smear is quick, economical, but easily and other mushrooms obscure, specificity and susceptibility are lower; Methods,anaerobic is the gold standard that present tetanus bacillus is identified, but high, the consuming time length of culture condition (about 18-72h) is often missed golden hour, causes serious consequence; Automatic bacterial culture identification appearance identifies that need carry out anaerobism to test sample equally cultivates, and has caused long shortcoming detection time thus; Foranalysis of nucleic acids mainly is to use fluorescent quantitative PCR technique, but existing round pcr exists number of drawbacks: (1) conditional request is high, and processes such as reagent preparation, sample pre-treatment, gene amplification and detection are accomplished in requirement respectively in three different operation intervals; (2) complicated operation need carry out a plurality of steps such as sample sex change, DNA extraction, pcr amplification, fluoroscopic examination.Need instruments such as water-bath, electric furnace, whizzer, super clean bench, amplification appearance in addition; (3) detection time long, 3-4h consuming time.Therefore seriously restricted should technology in clinical extensively carrying out, especially restricted in different medical unit, the other check of bed and the on-the-spot urgent application that detects.
Along with the continuous progress of molecular diagnostic techniques, some new foranalysis of nucleic acids technology are come out one after another, and loop-mediated isothermal amplification technology is exactly one of them.Though the loop-mediated isothermal amplification method has the short advantage of high specific, highly sensitive and proliferation time, need design primer voluntarily, the whether accurate specificity and the sensitivity that is related to detection of design of primers when using.In addition, the loop-mediated isothermal amplification method does not have supporting DNA rapid extracting method, and is still more numerous and diverse in the pre-treatment of sample, consuming time longer.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method and test kit thereof with loop-mediated isothermal amplification method detection tetanus bacillus, this detection method and test kit thereof are high, highly sensitive to the tetanus bacillus specificity, and testing process is simply quick.
The present invention provides a kind of method with loop-mediated isothermal amplification method detection tetanus bacillus, comprising:
Utilize loop-mediated isothermal amplification technology; With the tetanus bacillus sphacelotoxin gene in upper reaches inner primer, downstream inner primer, upper reaches outer primer and the downstream external primer amplification sample DNA; Said upper reaches inner primer has the nucleotide sequence shown in SEQ ID NO:1; Said downstream inner primer has the nucleotide sequence shown in SEQ ID NO:2; Said upper reaches outer primer has the nucleotide sequence shown in SEQ ID NO:3, and said downstream outer primer has the nucleotide sequence shown in SEQ ID NO:4;
Detect amplification.
The ultimate principle of loop-mediated isothermal amplification technology is the 2 pairs of special primers of 4 zone design (1 pair of inner primer and 1 pair of outer primer) to target gene; At constant temperature 63-67 ℃ of insulation 40-80min; Utilize high reactivity strand displacement archaeal dna polymerase (Bst archaeal dna polymerase); Make strand displacement DNA synthesize, thereby produce a large amount of stem ring-type amplified productions in ceaselessly oneself's circulation.These technological characteristics are: 1. high specific: to 4 kinds of special primers of 4 zone design of target gene, the RNA in 4 zones of non-specific binding is few simultaneously; 2. constant temperature, easy: amplification under constant temperature, easy and simple to handle; 3. highly sensitive: in 30-40min, can be expanded to 10 to target sequence
9-10
10Doubly, its lowest detection limit is about 10 copy/TEST.
The method of the invention is according to the ultimate principle of loop-mediated isothermal amplification technology; Adopting tetanus bacillus sphacelotoxin gene is target gene; To 2 pairs of special primers of its 4 zone design; Be upper reaches inner primer, downstream inner primer, upper reaches outer primer and downstream outer primer, because tetanus bacillus sphacelotoxin gene order is that variant serotype of tetanus bacillus and bacterial strain type are common, so can guarantee to detect the safety of the tetanus bacillus strain of different sources from the level of planting.Tetanus bacillus sphacelotoxin gene order derives from U.S. NCBI gene database (Clostridium tetani tetanus toxin gene; GenBank:AF154828.1), 4 kinds of special primers are used the primer-design software design that the Japanese Rong Yan Primerexplorer of company web site software designs and are obtained, and through U.S. NCBI gene database BLAST retrieval, satisfy specific requirement.
Sample DNA in the method for the invention extracts via following steps:
Sample and the abundant mixing of DNA rapid extraction liquid; Be heated to boiling and keep 1min; Centrifugal or filter, gained supernatant or filtrating are for comprising the solution of sample DNA, and said DNA rapid extraction liquid comprises the NaOH of 0.1-0.3mol/L and the Triton X-100 of 1.0-2.0%.Sample DNA utilizes DNA rapid extraction liquid according to the invention, can accomplish the extraction to tetanus bacillus DNA at 5-10min through the said extracted method, and the DNA that extracts can satisfy follow-up amplification requirement.
Aspect the detection amplification, can adopt agarose gel electrophoresis technology testing goal band, but the agarose gel electrophoresis technology required time is longer, be unfavorable for the rapid detection of tetanus bacillus.Event the present invention adopts the scattering turbidimetry method, changes through the turbidity of solution after the detecting instrument detection amplification and judges.As scheme more preferably, the present invention adopts colour developing liquid and amplified production effect generation colour-change to detect, and said colour developing liquid comprises fluorexon and MnCl
2When sample DNA did not increase, fluorexon combined with mn ion, showed filbert (feminine gender); A large amount of amplifications along with sample DNA produce pyrophosphate ion simultaneously, and pyrophosphate ion combines with mn ion, make fluorexon free, show yellow-green colour (positive).Like this, need not to pass through the unaided eye discrimination detected result by instrument.
When the method for the invention was provided, the present invention also provided a kind of test kit with loop-mediated isothermal amplification method detection tetanus bacillus, and said test kit comprises:
Upper reaches inner primer, downstream inner primer, upper reaches outer primer and downstream outer primer; Said upper reaches inner primer has the nucleotide sequence shown in SEQ ID NO:1; Said downstream inner primer has the nucleotide sequence shown in SEQ ID NO:2; Said upper reaches outer primer has the nucleotide sequence shown in SEQ ID NO:3, and said downstream outer primer has the nucleotide sequence shown in SEQ ID NO:4.
Wherein, upper reaches inner primer concentration is 6-10 μ mol/L, and downstream inner primer concentration is 6-10 μ mol/L, and upper reaches outer primer concentration is 0.5-1.5 μ mol/L, and downstream outer primer concentration is 0.5-1.5 μ mol/L.As preferably, upper reaches inner primer concentration is 8 μ mol/L, and downstream inner primer concentration is 8 μ mol/L, and upper reaches outer primer concentration is 1 μ mol/L, and downstream outer primer concentration is 1 μ mol/L.
As preferred version; Said test kit also comprises DNA rapid extraction liquid; Said DNA rapid extraction liquid comprises the NaOH of 0.1-0.3mol/L and the Triton X-100 of 1.0-2.0%; Preferably, said DNA rapid extraction liquid comprises the NaOH of 0.2mol/L and 1.25% Triton X-100, utilizes DNA rapid extraction liquid can realize the rapid extraction of sample DNA.
As further preferred version, said test kit also comprises constant-temperature amplification liquid, and said constant-temperature amplification liquid comprises the Tris-HCl of 20mmol/L pH8.8, the KCl of the 10mmol/L, (NH of 10mmol/L
4)
2SO
4, 2mmol/L MgS0
4, 0.1% Triton X-100, the dNTP of 40mmol/L, the Bst archaeal dna polymerase of 320U/mL.Wherein, the molar concentration rate of dTTP, dATP, dGTP, dCTP is 1: 1: 1 among the said dNTP: 1.
Scheme more preferably, said test kit also comprises colour developing liquid, and said colour developing liquid comprises 1.5-2.5mmol/L fluorexon and 2-4mmol/L MnCl
2, preferably, fluorexon concentration is 2mmol/L, MnCl
2Concentration is 3mmol/L.The by product magnesium pyrophosphate reaction that produces when colour developing liquid according to the invention and amplification tetanus bacillus sphacelotoxin gene; Fluorexon combines with mn ion during unreacted; It is filbert that solution is, and when pyrophosphate ion combines with mn ion, makes fluorexon free; Amplification back solution can be realized unaided eye discrimination tetanus bacillus detected result by the filbert yellow-green colour that changes into.
Can know that via above-mentioned technical scheme method and the test kit thereof with loop-mediated isothermal amplification method detection tetanus bacillus according to the invention is high, highly sensitive to the tetanus bacillus specificity, does not need specific apparatus, and be fast simple.
Embodiment
The invention discloses a kind of method and test kit thereof with loop-mediated isothermal amplification method detection tetanus bacillus, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.The method of the invention and test kit are described through preferred embodiment; The related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
According to the present invention, said method with loop-mediated isothermal amplification method detection tetanus bacillus comprises:
Utilize loop-mediated isothermal amplification technology; With the tetanus bacillus sphacelotoxin gene in upper reaches inner primer, downstream inner primer, upper reaches outer primer and the downstream external primer amplification sample DNA; Said upper reaches inner primer has the nucleotide sequence shown in SEQ ID NO:1; Said downstream inner primer has the nucleotide sequence shown in SEQ ID NO:2; Said upper reaches outer primer has the nucleotide sequence shown in SEQ ID NO:3, and said downstream outer primer has the nucleotide sequence shown in SEQ ID NO:4;
Detect amplification.
Wherein, said sample DNA extracts via following steps:
Sample and the abundant mixing of DNA rapid extraction liquid; Be heated to boiling and keep 1min; Centrifugal or filter, gained supernatant or filtrating are for comprising the solution of sample DNA, and said DNA rapid extraction liquid comprises the NaOH of 0.1-0.3mol/L and the Triton X-100 of 1.0-2.0%.
The present invention adopts the scattering turbidimetry method, changes through the turbidity of solution after the detecting instrument detection amplification and judges.As scheme more preferably, the present invention adopts colour developing liquid and amplification by product effect generation colour-change to detect, and said colour developing liquid comprises fluorexon and MnCl
2
The present invention also provides a kind of test kit with loop-mediated isothermal amplification method detection tetanus bacillus, and said test kit comprises:
Upper reaches inner primer, downstream inner primer, upper reaches outer primer and downstream outer primer; Said upper reaches inner primer has the nucleotide sequence shown in SEQ ID NO:1; Said downstream inner primer has the nucleotide sequence shown in SEQ ID NO:2; Said upper reaches outer primer has the nucleotide sequence shown in SEQ ID NO:3, and said downstream outer primer has the nucleotide sequence shown in SEQ ID NO:4.
Wherein, upper reaches inner primer concentration is 6-10 μ mol/L, and downstream inner primer concentration is 6-10 μ mol/L, and upper reaches outer primer concentration is 0.5-1.5 μ mol/L, and downstream outer primer concentration is 0.5-1.5 μ mol/L.As preferably, upper reaches inner primer concentration is 8 μ mol/L, and downstream inner primer concentration is 8 μ mol/L, and upper reaches outer primer concentration is 1 μ mol/L, and downstream outer primer concentration is 1 μ mol/L.
As preferred version; Said test kit also comprises DNA rapid extraction liquid; Said DNA rapid extraction liquid comprises the NaOH of 0.1-0.3mol/L and the Triton X-100 of 1.0-2.0%; Preferably, said DNA rapid extraction liquid comprises the NaOH of 0.2mol/L and 1.25% Triton X-100, utilizes DNA rapid extraction liquid can realize the rapid extraction of sample DNA.
As further preferred version, said test kit also comprises constant-temperature amplification liquid, and said constant-temperature amplification liquid comprises the Tris-HCl of 20mmol/L pH8.8, the KCl of the 10mmol/L, (NH of 10mmol/L
4)
2SO
4, 2mmol/L MgS0
4, 0.1% Triton X-100, the dNTP of 40mmol/L, the Bst archaeal dna polymerase of 320U/mL.
Scheme more preferably, said test kit also comprises colour developing liquid, and said colour developing liquid comprises 1.5-2.5mmol/L fluorexon and 2-4mmol/L MnCl
2, preferably, fluorexon concentration is 2mmol/L, MnCl
2Concentration is 3mmol/L.
The present invention adopts the tetanus bacillus reference culture to carry out feasibility Experiment, and it is positive to detect amplification with the scattering turbidimetry method, uses colour developing liquid detected result to be yellow-green colour, shows that the method for the invention and test kit thereof can be applicable to the detection of tetanus bacillus.
The method of the invention and test kit specificity experimental result thereof show, detect tetanus bacillus reference culture and clinical strains thereof with the scattering turbidimetry method, and the result all is positive, and all the other disturb bacterial strain all to be negative; Detect tetanus bacillus reference culture and clinical strains thereof with colour developing liquid, the result all is yellow-green colour, and all the other disturb bacterial strain all to be filbert.Show that test kit according to the invention and detection method thereof can carry out specific amplification to tetanus bacillus, during the wound infection anaerobism is cultivated in common interference bacterium and the common cultivation common interference bacterium all do not have amplification, specificity is preferably arranged.
The method of the invention and test kit sensitivity experiment result thereof show that tetanus bacillus reference culture minimal detectable concentration is 5x10
1CFU/uL shows that test kit according to the invention and detection method thereof have very high sensitivity.
The method of the invention and test kit degree of conformity experimental result thereof show; The sample of taking from the patient is all consistent with the result who detects through the full-automatic appearance of bacterium through the result that the method for the invention and test kit thereof detect; Shown that test kit according to the invention and detection method thereof have very high accuracy, can substitute the full-automatic appearance of bacterium and detect.
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: test kit according to the invention
1, test kit
The test kit of detection tetanus bacillus according to the invention comprises:
Upper reaches inner primer: have the nucleotide sequence shown in SEQ ID NO:1;
Downstream inner primer: have the nucleotide sequence shown in SEQ ID NO:2;
Upper reaches outer primer: have the nucleotide sequence shown in SEQ ID NO:3;
Downstream outer primer: have the nucleotide sequence shown in SEQ ID NO:4.
2, detection method
Add the reaction solution that comprises 2 μ L sample DNAs to reaction tubes; Add upper reaches inner primer, downstream inner primer, upper reaches outer primer and downstream outer primer again; 4 kinds of primer concentrations are respectively 8 μ mol/L, 8 μ mol/L, 1 μ mol/L and 1 μ mol/L; Mixing is positioned on the LA320C detecting instrument reacting hole 65 ℃ of constant-temperature amplification 60min.Change detected result according to reaction solution turbidity in the reaction tubes.
3, the method for the invention feasibility analysis
DNA with tetanus bacillus reference culture ATCC19406 extracts adopts above-mentioned detection method to detect, and positive control and negative control are set simultaneously.The tetanus type strain is available from being doctor Fluid Co., Ltd in the sky, Shanghai, positive control is provided by Japanese Rong Yan chemical company, and negative control is a saline water.
Detected result shows that positive control and tetanus bacillus reference culture detected result are positive, and the negative control detected result is negative, and shows that test kit of the present invention and detection method thereof are applicable to the detection of tetanus bacillus.
Embodiment 2: test kit according to the invention
1, test kit
The test kit of detection tetanus bacillus according to the invention comprises:
Upper reaches inner primer: have the nucleotide sequence shown in SEQ ID NO:1;
Downstream inner primer: have the nucleotide sequence shown in SEQ ID NO:2;
Upper reaches outer primer: have the nucleotide sequence shown in SEQ ID NO:3;
Downstream outer primer: have the nucleotide sequence shown in SEQ ID NO:4;
DNA rapid extraction liquid: the NaOH of 0.1mol/L and 1% Triton X-100.
2, detection method
Step 1: testing sample places aseptic throat swab, adds 1.0mL DNA rapid extraction liquid in the throat swab pipe, and mixing discards cotton swab,, keeps 1 minute to boiling with spirit lamp heating throat swab pipe;
Step 2: 4000 rev/mins of centrifugal 5min of whizzer, get supernatant to 1.5mL EP pipe, be template DNA to be checked; As do not have centrifugal condition, and available 2mL asepsis injector extracts throat swab pipe liquid, and through 0.2um needle-based bacterial filter, filtered solution is disposed in the 1.5mL EP pipe, is sample DNA.
Step 3: add the reaction solution that comprises 2 μ L sample DNAs to reaction tubes; Add upper reaches inner primer, downstream inner primer, upper reaches outer primer and downstream outer primer again; 4 kinds of primer concentrations are respectively 6 μ mol/L, 6 μ mol/L, 0.5 μ mol/L and 0.5 μ mol/L; Mixing is positioned on the LA320C detecting instrument reacting hole 65 ℃ of constant-temperature amplification 60min.Change detected result according to reaction solution turbidity in the reaction tubes.
3, the method for the invention feasibility analysis
Get the bacterium liquid 0.5mL of tetanus bacillus reference culture ATCC19406, bacterium liquid is diluted to 10
5CFU/uL adopts above-mentioned detection method to detect, and positive control and negative control are set simultaneously.The tetanus type strain is available from being doctor Fluid Co., Ltd in the sky, Shanghai, positive control is provided by Japanese Rong Yan chemical company, and negative control is a saline water.
Detected result shows that positive control and tetanus bacillus reference culture detected result are positive, and the negative control detected result is negative, and shows that test kit of the present invention and detection method thereof are applicable to the detection of tetanus bacillus.
Embodiment 3: test kit according to the invention
1, test kit
The test kit of detection tetanus bacillus according to the invention comprises:
Upper reaches inner primer: 10 μ mol/L have the nucleotide sequence shown in SEQ ID NO:1;
The downstream inner primer: 10 μ mol/L have the nucleotide sequence shown in SEQ ID NO:2;
Upper reaches outer primer: 1.5 μ mol/L have the nucleotide sequence shown in SEQ ID NO:3;
The downstream outer primer: 1.5 μ mol/L have the nucleotide sequence shown in SEQ ID NO:4;
DNA rapid extraction liquid: the NaOH of 0.3mol/L and 2% Triton X-100.
Constant-temperature amplification the liquid: (NH of the Tris-HCl of 20mmol/L pH8.8, the KCl of 10mmol/L, 10mmol/L
4)
2SO
4, 2mmol/L MgS0
4, 0.1% Triton X-100, the dNTP of 40mmol/L, the Bst archaeal dna polymerase of 320U/mL.
2, detection method
Step 1: testing sample places aseptic throat swab, adds 1.0mL DNA rapid extraction liquid in the throat swab pipe, and mixing discards cotton swab,, keeps 1 minute to boiling with spirit lamp heating throat swab pipe;
Step 2: 4000 rev/mins of centrifugal 5min of whizzer, get supernatant to 1.5mL EP pipe, be template DNA to be checked; As do not have centrifugal condition, and available 2mL asepsis injector extracts throat swab pipe liquid, and through 0.2um needle-based bacterial filter, filtered solution is disposed in the 1.5mL EP pipe, is sample DNA.
Step 3: add 2 μ L sample DNAs and 23 μ L constant-temperature amplification liquid to reaction tubes; Add upper reaches inner primer, downstream inner primer, upper reaches outer primer and downstream outer primer again; 4 kinds of primer concentrations are respectively 6 μ mol/L, 6 μ mol/L, 0.5 μ mol/L and 0.5 μ mol/L; Mixing is positioned on the LA320C detecting instrument reacting hole 65 ℃ of constant-temperature amplification 60min.Change detected result according to reaction solution turbidity in the reaction tubes.
3, the method for the invention feasibility analysis
Get the bacterium liquid 0.5mL of tetanus bacillus reference culture ATCC19406, bacterium liquid is diluted to 10
5CFU/uL adopts above-mentioned detection method to detect, and positive control and negative control are set simultaneously.The tetanus type strain is available from being doctor Fluid Co., Ltd in the sky, Shanghai, positive control is provided by Japanese Rong Yan chemical company, and negative control is a saline water.
Detected result shows that positive control and tetanus bacillus reference culture detected result are positive, and the negative control detected result is negative, and shows that test kit of the present invention and detection method thereof are applicable to the detection of tetanus bacillus.
Embodiment 4: test kit according to the invention
1, test kit
The test kit of detection tetanus bacillus according to the invention comprises:
Upper reaches inner primer: 8 μ mol/L have the nucleotide sequence shown in SEQ ID NO:1;
The downstream inner primer: 8 μ mol/L have the nucleotide sequence shown in SEQ ID NO:2;
Upper reaches outer primer: 1 μ mol/L has the nucleotide sequence shown in SEQ ID NO:3;
The downstream outer primer: 1 μ mol/L has the nucleotide sequence shown in SEQ ID NO:4;
DNA rapid extraction liquid: the NaOH of 0.2mol/L and 1.25% Triton X-100;
Constant-temperature amplification the liquid: (NH of the Tris-HCl of 20mmol/L pH8.8, the KCl of 10mmol/L, 10mmol/L
4)
2SO
4, 2mmol/L MgS0
4, 0.1% Triton X-100, the dNTP of 40mmol/L, Bst archaeal dna polymerase, 2mmol/L fluorexon and the 3mmol/L MnCl of 320U/mL
2
2, detection method
Step 1: testing sample places aseptic throat swab, adds 1.0ml DNA rapid extraction liquid in the throat swab pipe, and mixing discards cotton swab,, keeps 1 minute to boiling with spirit lamp heating throat swab pipe;
Step 2: 4000 rev/mins of centrifugal 5min of whizzer, get supernatant to 1.5mL EP pipe, be template DNA to be checked; As do not have centrifugal condition, and available 2mL asepsis injector extracts throat swab pipe liquid, and through 0.2um needle-based bacterial filter, filtered solution is disposed in the 1.5mL EP pipe, is sample DNA.
Step 3: add 2 μ L sample DNAs and 23 μ L constant-temperature amplification liquid to reaction tubes; Add upper reaches inner primer, downstream inner primer, upper reaches outer primer and downstream outer primer again; 4 kinds of primer concentrations are respectively 6 μ mol/L, 6 μ mol/L, 0.5 μ mol/L and 0.5 μ mol/L; Mixing is positioned on the LA320C detecting instrument reacting hole 65 ℃ of constant-temperature amplification 60min.According to reaction solution colour-change in the reaction tubes, detected result.
3, the method for the invention feasibility analysis
Get the bacterium liquid 0.5mL of tetanus bacillus reference culture ATCC19406, bacterium liquid is diluted to 10
5CFU/uL adopts above-mentioned detection method to detect, and positive control and negative control are set simultaneously.The tetanus type strain is available from being doctor Fluid Co., Ltd in the sky, Shanghai, positive control is provided by Japanese Rong Yan chemical company, and negative control is a saline water.
Detected result shows that positive control and tetanus bacillus reference culture detected result are yellow-green colour for the reaction solution color, and the negative control detected result shows that for the reaction solution color is filbert test kit of the present invention and detection method thereof are applicable to the detection of tetanus bacillus.
Embodiment 5: the method for the invention and test kit specificity analyses thereof
Get the bacterium liquid 0.5mL of tetanus bacillus reference culture, tetanus bacillus clinical strains, interference bacterium reference culture and interference bacterium clinical strains respectively, bacterium liquid is diluted to 10
5CFU/uL adopts embodiment 1 to embodiment 4 said method and test kit to detect.The secretory product anaerobism that clinical strains infects by open injury patient deep wound is cultivated and is cultivated the appearance evaluation by the VITEK-2 automatic bacterial and obtains; All available from be doctor Fluid Co., Ltd in the sky, Shanghai, each strain name and numbering are referring to table 1 for reference culture, and detected result is referring to table 2.
Table 1 strain name and numbering
| Strain name | Strain number and source | Strain name | Strain number and source |
| Tetanus bacillus | ATCC19406 | Clostridium butylicum | Clinical strains |
| Tetanus bacillus | Clinical strains | Clostridium bifermentans | Clinical strains |
| Clostridium perfringens | ATCC13124 | Clostridium tertium | Clinical strains |
| Clostridium perfringens | Clinical strains | Bifidus bacillus | Clinical strains |
| Difficile toxins | ATCC9689 | Streptococcus aureus | ATCC25923 |
| Clostridium novyi | ATCC19402 | Escherichia coli | ATCC25922 |
| Clostridium histolyticum | ATCC19401 | Pseudomonas aeruginosa | ATCC27853 |
| Clostridium histolyticum | Clinical strains | Streptococcus pneumoniae | ATCC49619 |
| Clostridium relieves internal heat | Clinical strains |
Table 2 specificity analyses detected result
The result shows, detects tetanus bacillus reference culture and clinical strains thereof with the scattering turbidimetry method, and the result all is positive, and all the other disturb bacterial strain all to be negative; Detect tetanus bacillus reference culture and clinical strains thereof with colour developing liquid; The result all is yellow-green colour; And all the other disturb bacterial strain all to be filbert; Show that test kit according to the invention and detection method thereof can carry out specific amplification to tetanus bacillus, during the wound infection anaerobism is cultivated in common interference bacterium and the common cultivation common interference bacterium all do not have amplification, specificity is preferably arranged.
Embodiment 6: the method for the invention and test kit sensitivity analysis thereof
Tetanus bacillus reference culture ATCC19406 is seeded in 37 ℃ of anaerobism cultivation 48-72h on the high density agar anaerobism blood agar, counting bacterium colony.According to colony count, dilute bacterium liquid to 5x10 with the saline water adjustment
7CFU/uL, 5x10
6CFU/uL, 5x10
5CFU/uL, 5x10
4CFU/uL, 5x10
3CFU/uL, 5x10
2CFU/uL, 5x10
1CFU/uL, 5x10
0CFU/uL.Reference culture ATCC19406 is available from be doctor Fluid Co., Ltd in the sky, Shanghai.
Get the tetanus bacillus bacterium liquid 0.5mL of each concentration, adopt embodiment 1 to embodiment 4 said method and test kit thereof to detect, detected result is referring to table 3.
Table 3 sensitivity analysis detected result
| Bacterial concentration | Detected result | Bacterial concentration | Detected result |
| 5x10 7CFU/uL | +/yellow-green colour | 5x10 3CFU/uL | +/yellow-green colour |
| 5x10 6CFU/uL | +/yellow-green colour | 5x10 2CFU/uL | +/yellow-green colour |
| 5x10 5CFU/uL | +/yellow-green colour | 5x10 1CCFFUU//uuLL | +/yellow-green colour |
| 5x10 4CFU/uL | +/yellow-green colour | 5x10 0CFU/uL | -/filbert |
Shown in the result, detect tetanus bacillus reference culture 5x10 with the scattering turbidimetry method
1-5x10
7Each concentration detected result of CFU/uL is all positive, and 5x10
0CFU/uL is negative; Detect tetanus bacillus reference culture 5x10 with colour developing liquid
1-5x10
7Each concentration detected result of CFU/uL is yellow-green colour, and 5x10
0CFU/uL is filbert.The result shows that the peak response that the present invention detects tetanus bacillus is 5x10
1CFU/uL.
Embodiment 7: the method for the invention and test kit degree of conformity thereof are analyzed
Get the secretory product that 16 open injury patient deep wounds infect, adopt embodiment 1 to embodiment 4 said method and test kit thereof to detect, adopt the VITEK-2 automatic bacterial to cultivate simultaneously and identify that the result is referring to table 4 after the appearance anaerobism is cultivated.
Table 4 degree of conformity analyzing and testing result
| The sample numbering | VITEK-2 identifies | Constant-temperature amplification |
| 1 | Tetanus bacillus | +/yellow-green colour |
| 2 | Tetanus bacillus | +/yellow-green colour |
| 3 | Tetanus bacillus | +/yellow-green colour |
| 4 | Tetanus bacillus | +/yellow-green colour |
| 5 | Clostridium histolyticum | -/filbert |
| 6 | Bifidus bacillus | -/filbert |
| 7 | Clostridium perfringens | -/filbert |
| 8 | Clostridium bifermentans | -/filbert |
| 9 | Clostridium perfringens | -/filbert |
| 10 | Clostridium tertium | -/filbert |
| 11 | Clostridium perfringens | -/filbert |
| 12 | Clostridium perfringens | -/filbert |
| 13 | Clostridium perfringens | -/filbert |
| 14 | Clostridium perfringens | -/filbert |
| 15 | Clostridium butylicum | -/filbert |
| 16 | Clostridium perfringens | -/filbert |
The result shows; The VITEK-2 automatic bacterial is cultivated the tetanus bacillus that appearance identifies; Its constant-temperature amplification is the result all be positive or yellow-green colour; And other bacterial strains that identify all are negative or are filbert, and it is consistent to show that test kit according to the invention and detection method thereof and automatic bacterial are cultivated appearance culture identification result, has very high degree of conformity.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (5)
1. the test kit with loop-mediated isothermal amplification method detection tetanus bacillus is characterized in that, comprising:
Upper reaches inner primer, downstream inner primer, upper reaches outer primer and downstream outer primer; Said upper reaches inner primer is made up of the nucleotide sequence shown in SEQ ID NO:1; Said downstream inner primer is made up of the nucleotide sequence shown in SEQ ID NO:2; Said upper reaches outer primer is made up of the nucleotide sequence shown in SEQ ID NO:3, and said downstream outer primer is made up of the nucleotide sequence shown in SEQ ID NO:4.
2. according to the said test kit of claim 1, it is characterized in that, also comprise DNA rapid extraction liquid, said DNA rapid extraction liquid comprises the NaOH of 0.1-0.3mol/L and the Triton X-100 of 1.0-2.0%.
3. according to the said test kit of claim 1, it is characterized in that, also comprise constant-temperature amplification liquid, said constant-temperature amplification liquid comprises the Tris-HCl of 20mmol/L pH8.8, the KCl of the 10mmol/L, (NH of 10mmol/L
4)
2SO
4, 2mmol/L MgS0
4, 0.1% Triton X-100, the dNTP of 40mmol/L, the Bst archaeal dna polymerase of 320U/mL.
4. according to the said test kit of claim 2, it is characterized in that said DNA rapid extraction liquid comprises the NaOH of 0.2mol/L and 1.25% Triton X-100.
5. according to the said test kit of claim 3, it is characterized in that, also comprise colour developing liquid, said colour developing liquid comprises 1.5-2.5mmol/L fluorexon and 2-4mmol/L MnCl
2
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101082579A (en) * | 2006-05-29 | 2007-12-05 | 广州华峰生物科技有限公司 | Gene rapid diagnosis method based on annular mediated isothermal amplification technology |
| CN101302553A (en) * | 2008-05-30 | 2008-11-12 | 广州华峰生物科技有限公司 | Mycobacterium tuberculosis gene rapid diagnosis reagent kit base on loop-mediated isothermal amplification technology and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101082579A (en) * | 2006-05-29 | 2007-12-05 | 广州华峰生物科技有限公司 | Gene rapid diagnosis method based on annular mediated isothermal amplification technology |
| CN101302553A (en) * | 2008-05-30 | 2008-11-12 | 广州华峰生物科技有限公司 | Mycobacterium tuberculosis gene rapid diagnosis reagent kit base on loop-mediated isothermal amplification technology and detection method thereof |
Non-Patent Citations (1)
| Title |
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| 张体银 等.环介导等温扩增技术快速检测食品中的单增李斯特氏菌.《中国食品学报》.2010,第10卷(第3期),200-204. * |
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