CN109750114A - The constant-temperature amplification detection method and its primer special and kit of the sour klebsiella spp of production - Google Patents
The constant-temperature amplification detection method and its primer special and kit of the sour klebsiella spp of production Download PDFInfo
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Abstract
The invention discloses the constant-temperature amplification detection method for producing sour klebsiella spp and its primer specials and kit.The present invention provides for detecting the complete reaction primer for producing the ring mediated isothermal amplification of sour klebsiella spp, it is made of primers F 3, primer B3, primers F IP, primer BIP, primer LoopF and primer LoopB；The primers F 3, primer B3, primers F IP, primer BIP, primer LoopF and the corresponding nucleic acid sequence of primer LoopB are respectively sequence 1 in sequence table to sequence 6.Primer of the present invention can under isothermal conditions quickly, conveniently, efficiently, Gao Teyi, detection produces sour klebsiella spp with sensitivity, complex instrument is not needed, new technology platform is provided to produce the detection of sour klebsiella spp, it can be used for primary care health unit and disease prevention and control center's screening and detection produce sour klebsiella spp, have a vast market foreground with biggish economical, societal benefits, be suitable for a wide range of promote and apply.
Description
Technical field
The invention belongs to field of biotechnology more particularly to a kind of constant-temperature amplification detection method for producing sour klebsiella spp and
Its primer special and kit.
Background technique
Klebsiella spp is Nosocomial Infections and the important pathogen of nosocomial infection, especially in immune deficient patients and ICU weight
Disease patient can cause serious pulmonary infection and bloodstream infection, have very high morbidity and mortality.Klebsiella
Mainly include klebsiella pneumoniae, produce sour klebsiella spp, solution ornithine klebsiella spp, plant raw klebsiella spp and autochthonal
5 strains of klebsiella spp, wherein klebsiella pneumoniae and the sour klebsiella spp of production are to cause people's pulmonary infection and blood flow sense
The important pathogen body of dye.In addition to causing pulmonary infection and bloodstream infection, producing sour klebsiella spp is also food poisoning and antibiotic
The important pathogen body of related hemorrhages colitis.New England's magazine is non-clostridium difficile there are some researches prove sour klebsiella spp is produced
The main pathogenic fungi of caused antibiotic related hemorrhages colitis.Producing sour klebsiella spp can have with secretory cell toxin
Very strong virulence and very high pathogenic has research to confirm that after mouse peritoneal is injected and produces sour klebsiella spp, experiment mice exists
For 24 hours all because infection is dead.The detection method of the sour klebsiella spp of traditional production, including the experiment of lactic acid bacteria degradation analysis, pine three
Sugar is typically based on Phenotypic examination, therefore be difficult to produce using experiment, API identification method and the automatic identification method of VITEK2 bacterium etc.
Sour klebsiella spp mutually identifies with the bacterium of other Klebsiellas such as klebsiella pneumoniae.Currently used molecular gene
Classifying method, such as pulsed field gel electrophoresis, Analysis of random amplified polymorphic DNA and amplified fragment length polymorphism, it is right
Experimental facilities and operator have very high requirement, are unsuitable for basic medical unit popularization.Therefore, need to establish one kind at present
Quickly, easy, economic, efficient, special production acid klebsiella spp detection method.
Ring mediated isothermal amplification method (Loop-Mediated Isothermal Amplification, LAMP) is a kind of new
Type constant temperature DNA rapid amplifying technology, under the effect of Bst archaeal dna polymerase, 65 DEG C of constant temperature are realized to the efficient, fast of target-gene sequence
The special amplification of speed, specificity.Compared with traditional round pcr, LAMP requires experimental facilities low, it is only necessary to a common water-bath
Can meet the requirements, and be added color developing agent after, reaction result naked eyes as it can be seen that can be used for the quick diagnosis of infectious diseases with
And the epidemiological surveillance of the pathogen infections such as bacterium, virus and helminth.
Summary of the invention
It is an object of the present invention to provide the complete anti-of the ring mediated isothermal amplification for detecting the sour klebsiella spp of production
Answer primer.
Primer set provided by the invention, by primers F 3, primer B3, primers F IP, primer BIP, primer LoopF and primer
LoopB composition；
The primers F 3 is following (a1) or (a2):
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) sequence 1 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1
The DNA molecular of identical function；
The primer B3 is following (a3) or (a4):
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) sequence 2 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2
The DNA molecular of identical function；
The primers F IP is following (a5) or (a6):
(a5) single strand dna shown in the sequence 3 of sequence table；
(a6) sequence 3 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3
The DNA molecular of identical function；
The primer BIP is following (a7) or (a8):
(a7) single strand dna shown in the sequence 4 of sequence table；
(a8) sequence 4 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4
The DNA molecular of identical function；
The primer LoopF is following (a9) or (a10):
(a9) single strand dna shown in the sequence 5 of sequence table；
(a10) sequence 5 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5
The DNA molecular of identical function；
The primer LoopB is following (a11) or (a12):
(a11) single strand dna shown in the sequence 6 of sequence table；
(a12) sequence 6 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5
The DNA molecular of identical function.
In above-mentioned primer, the primers F 3, the primer B3, the primers F IP, the primer BIP, the primer
The molar ratio of LoopF and the primer LoopB are 1:1:8:8:4:4.
Another object of the present invention is to provide for detecting the loop-mediated isothermal amplification reaction reagent for producing sour klebsiella spp.
Reaction reagent provided by the invention, including above-mentioned primer set.
Mentioned reagent further includes archaeal dna polymerase, dNTP, Tris-HCl, MgSO4、KCl、(NH4)2SO4, glycine betaine and
Tween-20。
Above-mentioned loop-mediated isothermal amplification reaction reagent is by archaeal dna polymerase, dNTP, Tris-HCl, MgSO4、KCl、(NH4)2SO4, glycine betaine, Tween-20 and above-mentioned primer and water composition.
The primers F 3, the primer B3, the primers F IP, the primer BIP, the primer LoopF and the primer
The concentration of LoopB is respectively 0.2 μM, 0.2 μM, 1.6 μM, 1.6 μM, 0.8 μM and 0.8 μM.
Third purpose of the present invention is to provide for detecting the loop-mediated isothermal amplification kit for producing sour klebsiella spp.
Kit provided by the invention comprising above-mentioned primer or above-mentioned ring Jie's isothermal duplication reagent.
For convenience of detection, it may also include positive control and negative control in the kit, the positive control is to produce acid
The DNA of klebsiella spp reference culture K.oxytoca ATCC 700324, the negative control are the LAMP amplification without DNA
System (such as distilled water).
Specifically, the LAMP detection kit of the production acid klebsiella spp be divided into real-time nephelometry detection kit and
calcein/Mn2+2 kinds of fluorescence developing method detection kit.Real-time nephelometry LAMP detection kit detection reagent volume is 25 μ
L, by 1.4mM dNTP, 20mM Tris-HCl (pH 8.8), 8mM MgSO4, 10mM KCl, 10mM (NH4)2SO4, 0.8M beet
Alkali, 0.1%Tween-20,8U BstDNA polymerase, 0.2 μM of positive outer primer KO-16F3,0.2 μM of reversed outer primer KO-
16B3,1.6 μM of positive inner primer KO-16FIP, 1.6 μM of reversed inner primer KO-16BIP, 0.8 μM of positive ring primer KO-
16LoopF, 0.8 μM of reversed ring primer KO-16LoopB and water composition.
calcein/Mn2+1 μ l is added on the basis of the reaction system of real-time nephelometry kit in fluorescence developing method kit
Calcein/Mn2+Fluorescence indicator, calcein/Mn2+Fluorescence indicator is by 1.3mM calcein and 26mM MnCl2It is dissolved in
70% (volumn concentration) dimethyl sulfoxide is formulated.calcein/Mn2+The detection examination of fluorescence developing method LAMP detection kit
Agent volume is 26 μ l, by 1.4mM dNTP, 20mM Tris-HCl (pH 8.8), 8mM MgSO4, 10mM KCl, 10mM (NH4)2SO4, 0.8M glycine betaine, 0.1%Tween-20,8U BstDNA polymerase, 0.2 μM of positive outer primer KO-16F3,0.2 μM reversed
Outer primer KO-16B3,1.6 μM of positive inner primer KO-16FIP, 1.6 μM of reversed inner primer KO-16BIP, 0.8 μM of positive ring primer
KO-16LoopF, 0.8 μM of reversed ring primer KO-16LoopB, the calcein/Mn of 1 μ l2+Fluorescence indicator and water composition.
Above-mentioned primer set or above-mentioned PCR reagent it is following 1) or 2) or 3) in application be also model that the present invention protects
It encloses:
1) preparation identification or auxiliary identification produce the product of sour klebsiella spp；
2) whether contain the product for producing sour klebsiella spp in preparation detection sample to be tested.
3) preparation detection or auxiliary detect whether bacterium to be measured is the product for producing sour klebsiella spp.
In above-mentioned application, the product is kit.
4th purpose of the invention is to provide a kind of method identified or auxiliary identification produces sour klebsiella spp.
Method provided by the invention, includes the following steps:
(1) total DNA of sample to be tested is extracted；
(2) total DNA extracted using step (1) carries out ring mediated isothermal amplification using above-mentioned primer sets as template；
Judged with following A or B:
A, with transmissometer detect amplified production, if amplified production turbidity in S curve variation, the tested microorganism be or
Candidate is the sour klebsiella spp of production；If amplified production turbidity does not change, the tested microorganism is not or candidate is not
Produce sour klebsiella spp；
B, the fluorescence indicator is added in ring mediated isothermal amplification forward reaction system, observes after reaction,
If amplified production color changes, the tested microorganism is or candidate is to produce sour klebsiella spp；
If amplified production color does not change, the tested microorganism is not or candidate is not to produce the primary bar of sour Cray
Bacterium.
5th purpose of the invention be to provide a kind of detection or auxiliary detection in sample whether containing producing the primary bar of sour Cray
The method of bacterium.
Method provided by the invention, includes the following steps:
(1) total DNA of sample to be tested is extracted；
(2) total DNA extracted using step (1) carries out ring mediated isothermal amplification using above-mentioned primer sets as template；
Judged with following A or B:
A, amplified production is detected with transmissometer, if reaction solution before and after S curve or ring mediated isothermal amplification occurs in amplified production
Turbidity rise, then in sample to be detected containing producing sour klebsiella spp；If amplified production does not occur S curve or ring mediated isothermal
The turbidity of amplification front and back reaction solution does not change, then without containing the sour klebsiella spp of production in the sample to be detected；
B, the fluorescence indicator is added in ring mediated isothermal amplification forward reaction system, observes after reaction,
If amplified production color changes, the sour klebsiella spp of production is contained in the sample to be detected；
If amplified production color does not change, without containing the sour klebsiella spp of production in the sample to be detected.
Above-mentioned amplified production color changes, and for amplified production, color changes compared with expanding preceding reaction solution, at this
In the embodiment of invention, amplified production color, which changes, becomes green for amplified production color；
Above-mentioned amplified production color does not change, and for amplified production, color does not change compared with expanding preceding reaction solution,
In an embodiment of the present invention, it is still orange that amplified production color, which does not change as amplified production color,；
In the above method, the temperature of the LAMP reaction is 65 DEG C.
In the above method, the template of the LAMP reaction is the genomic DNA of sample to be detected.
Above scheme is used, the invention has the following advantages that
1) high specific: 6 specific primers identify 8 specific regions for producing sour klebsiella spp target sequence,
It ensure that the high degree of specificity of LAMP amplification.
2) highly sensitive: remolding sensitivity regular-PCR method is 10 times high；
3) result identification is easy: can observe by the naked eye result (calcein/Mn2+Fluorescence developing method), it can also directly use
Transmissometer judging result；
4) easy to operate: as long as will test the DNA of sample extraction and detection reagent is put into togerther in 65 DEG C of thermostat water baths
After sixty minutes it may determine that result；
5) it quick, efficient amplification: can be completed in entire LAMP amplified reaction one hour, and detectable limit can reach
4.8pg/μl。
In conclusion LAMP primer of the invention can under isothermal conditions quickly, conveniently, efficiently, it is Gao Teyi, highly sensitive
Ground detection produces sour klebsiella spp, does not need complex instrument, provides new technology platform to produce the detection of sour klebsiella spp,
It can be used for primary care health unit and disease prevention and control center's screening and detection produce sour klebsiella spp, there is wide city
Field prospect and biggish economical, societal benefits are suitable for a wide range of promote and apply.
The present invention is described in further details combined with specific embodiments below.
Detailed description of the invention
Fig. 1 is the transmissometer result that the present invention produces the best primer of sour klebsiella spp LAMP detection method.
Fig. 2 is the specific detection result that the present invention produces the real-time turbidity LAMP detection method of sour klebsiella spp.
Fig. 3 is that the present invention produces sour klebsiella spp calcein/Mn2+The specific detection knot of fluorescence developing LAMP detection method
Fruit.
Fig. 4 is the sensitivity technique result that the present invention produces the real-time turbidity LAMP detection method of sour klebsiella spp.
Fig. 5 is that the present invention produces sour klebsiella spp calcein/Mn2+The sensitivity technique knot of fluorescence developing LAMP detection method
Fruit.
Fig. 6 is to produce sour klebsiella spp Standard PCR detection method sensitivity technique result.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1, the design of primers for carrying out LAMP detection to the sour klebsiella spp of production
One, for the design of primers for producing sour klebsiella spp progress LAMP detection
From U.S. gene database GenBank retrieval obtain the sour klebsiella spp sequence of production (No. GenBank:
AY065648.1), it is designed for carrying out LAMP detection to the sour klebsiella spp of production with software Primer Explorer Ver.5
5 sets of primer combinations, respectively KO-l, KO-5, KO-9, KO-13 and KO-16, each primer sequence such as the following table 1:
Table 1 is that LAMP detects alternative primer
Two, the present invention produces the foundation of the LAMP detection method of sour klebsiella spp and the screening of best primer
The 5 sets of primer pairs for carrying out LAMP detection to the sour klebsiella spp of production obtained with above-mentioned one produce the primary bar of sour Cray
Bacterium reference culture K.oxytoca ATCC 700324 carries out LAMP detection, same reaction conditions (setting 65 DEG C of constant temperature 60min)
Under, different primers are added in the reaction system and combine KO-l, KO-5, KO-9, KO-13, KO-16, to determine optimum response primer.
The specific method is as follows:
Template preparation: it is extracted with the Wizard Genomic DNA purification Kit commercialization DNA of Promega
Kit extracts the genomic DNA for producing sour klebsiella spp；
Real-time nephelometry LAMP reaction system: total volume is 25 μ l, wherein containing 1.4mM dNTPs, 20mM Tris-HCl
(pH 8.8)、8mM MgSO4、10mM KCl、10mM(NH4)2SO4, 0.8M glycine betaine, 0.1% (volumn concentration) tween-
20,8U Bst archaeal dna polymerases, 0.2 μM of F3,0.2 μM of B3,1.6 μM of FIP, 1.6 μM, 1.6 μM of BIP, 0.8 μM of LoopF,
0.8 μM of LoopB, 2 μ, 1 genomic DNA, supply volume with water.
Above-mentioned LAMP reaction condition: 60min in 65 DEG C of thermostat water baths is set.
Real-time nephelometry LAMP reaction system carries out LAMP reaction, directly detects reaction front and back with transmissometer after reaction
The turbidity variation of reaction solution carrys out judging result (principle are as follows: LAMP can generate magnesium pyrophosphate during reacting, and magnesium pyrophosphate is one
Kind white precipitate, transmissometer can change judge that LAMP is reacted according to real-time turbidity), it is then sun that turbidity variation, which is in S curve,
Property, it indicates to be not present in the unchanged expression sample to be tested of turbidity containing sour klebsiella spp (positive) is produced in sample to be tested and produce acid
Klebsiella spp (feminine gender).
As a result as shown in Figure 1, KO-l, KO-5, KO-9, KO-13, KO-16 are the different primer combination of table 1, wherein KO-16
Appearance time is earliest, therefore selects to produce the best primer of acid klebsiella spp LAMP detection.
Therefore, KO-16 group primer (sequence 1-6 in sequence table) is best LAMP detection primer in table 1.
Three, the present invention produces the LAMP detection kit of sour klebsiella spp
The LAMP detection kit for producing sour klebsiella spp includes primer shown in sequence 1-6 in sequence table.Or, producing acid gram
The LAMP detection kit of the primary bacillus of thunder includes following LAMP reagent As or LAMP reagent B:
LAMP reagent A is by 1.4mM dNTPs, 20mM Tris-HCl (pH 8.8), 8mM MgSO4、10mM KCl、10mM
(NH4)2SO4, 0.8M glycine betaine, 0.1% (volumn concentration) Tween-20,8U Bst archaeal dna polymerase, 0.2 μM of KO-
16F3,0.2 μM of KO-16B3,1.6 μM of KO-16FIP, 1.6 μM of KO-16BIP, 0.8 μM of KO-16LoopF and 0.8 μM of KO-
16LoopB and water composition；
Or, LAMP reagent B includes the calcein/Mn of above-mentioned LAMP reagent A and 1 μ l2+Fluorescence indicator；calcein/Mn2 +Fluorescence indicator is by 1.3mM calcein and 26mM MnCl270% dimethyl sulfoxide is dissolved in be formulated.
Mentioned reagent box can also include the production acid klebsiella spp K.oxytoca ATCC as positive control
700324 genomic DNA and the LAMP amplification system (distilled water) without DNA as negative control.
Embodiment 2, the present invention produce the specificity of the LAMP detection method of sour klebsiella spp, sensitivity detection
One, the present invention produces the specific detection of the LAMP detection method of sour klebsiella spp
1, LAMP reacts
The genomic DNA of following bacterial strain is extracted respectively:
1,K.oxytoca ATCC 700324；2,Klebsiella pneumoniae ATCC 2146；3,
Klebsiella pneumoniae ATCC 7105；4,Klebsiella rhinoscleromatis CMCC46111；5,
Haemophilus influenza ATCC49247；6,Salmonella typhi 9275；7,Streptococcus
pneumoniae 112-07；8,Escherichia coli 44825；9,Pseudomonas aeruginosa ATCC
15442；10,Shigella flexneri4536；11,Legionella pneumophila 9135；12,Proteus
vulgaris CMCC49027；13,Mycobacterium tuberculosis 005；14,Acinetobacter
baumannii 12101；15,Stenotrophomonas maltophilia K279a；16,negative control
(double-distilled water).
1) real-time turbidity LAMP method
Real-time turbidity LAMP method reaction system: total volume is 25 μ l, wherein containing 1.4mM dNTPs, 20mM Tris-HCl
(pH 8.8)、8mM MgSO4、10mM KCl、10mM(NH4)2SO4, 0.8M glycine betaine, 0.1% (volumn concentration) tween-
20,8U Bst archaeal dna polymerases, 0.2 μM of KO-16F3,0.2 μM of KO-16B3,1.6 μM of KO-16FIP, 1.6 μM of KO-
16BIP, 0.8 μM of KO-16LoopF, 0.8 μM of KO-16LoopB, 2 μ, 1 genomic DNA, supply volume with water.
LAMP reaction condition are as follows: 65 DEG C, constant temperature 60min.
Real-time nephelometry LAMP reaction system carries out LAMP reaction, directly detects reaction front and back with transmissometer after reaction
The turbidity variation of reaction solution carrys out judging result (principle are as follows: LAMP can generate magnesium pyrophosphate during reacting, and magnesium pyrophosphate is one
Kind white precipitate, transmissometer can change judge that LAMP is reacted according to real-time turbidity), it is then sun that turbidity variation, which is in S curve,
Property, it indicates to react containing sour klebsiella spp (positive) is produced in the unchanged expression sample to be tested of turbidity of front and back not in sample to be tested
In the presence of the sour klebsiella spp (feminine gender) of production.
As a result as shown in Fig. 2, 1, K.oxytoca ATCC 700324；2,Klebsiella pneumoniae
ATCC2146；3,Klebsiella pneumoniae ATCC 7105；4,Klebsiella rhinoscleromatis
CMCC46111；5,Haemophilus influenza ATCC49247；6,Salmonella typhi 9275；7,
Streptococcus pneumoniae 112-07；8,Escherichia coli 44825；9,Pseudomonas
aeruginosa ATCC 15442；10,Shigella flexneri 4536；11,Legionella pneumophila
9135；12,Proteus vulgaris CMCC49027；13,Mycobacterium tuberculosis 005；14,
Acinetobacter baumannii 12101；15,Stenotrophomonas maltophilia K279a；16,
Negative control (double-distilled water), it can be seen that the LAMP detection method is only producing sour Cray
The sigmoid curve for representing positive reaction is generated in primary bacillus positive sample K.oxytoca ATCC 700324, illustrates the detection method
With high specific.
2)calcein/Mn2+Fluorescence developing LAMP method
The calcein/Mn of 1 μ l is added in real-time turbidity LAMP method reaction system before LAMP reaction2+Fluorescence indicator.
calcein/Mn2+Fluorescence indicator is dissolved in 70% dimethyl sulfoxide by 1.3mM calcein and 26mM MnCl2 and is formulated.
calcein/Mn2+The total volume of fluorescence developing method LAMP reaction is 26 μ l, wherein contain tetra- kinds of dNTP of 1.4mM,
20mM Tris-HCl (pH 8.8), 8mM MgSO4, 10mM KCl, 10mM (NH4)2SO4, 0.8M glycine betaine, 0.1% tween-
20,8U Bst archaeal dna polymerases, 0.2 μM of KO-16F3 and KO-16B3,1.6 μM of KO-16FIP and KO-16BIP, 0.8 μM of KO-
16LoopF and KO-16LoopB, the calcein/Mn of 1 μ l2+Fluorescence indicator and the sample DNA to be detected of 2 μ 1, supply body with water
Product.
LAMP reaction condition are as follows: 65 DEG C, constant temperature 60min.
If LAMP reaction product becomes green, then it represents that containing the sour klebsiella spp (positive) of production in sample to be tested, if
LAMP reaction product is still orange, then it represents that there is no produce sour klebsiella spp (feminine gender) in sample to be tested.
As a result as shown in figure 3,1, K.oxytoca ATCC 700324；2,Klebsiella pneumoniae ATCC
2146；3,Klebsiella pneumoniae ATCC 7105；4,Klebsiella rhinoscleromatis
CMCC46111；5,Haemophilus influenza ATCC49247；6,Salmonella typhi 9275；7,
Streptococcus pneumoniae 112-07；8,Escherichia coli 44825；9,Pseudomonas
aeruginosa ATCC 15442；10,Shigella flexneri 4536；11,Legionella pneumophila
9135；12,Proteus vulgaris CMCC49027；13,Mycobacterium tuberculosis 005；14,
Acinetobacter baumannii 12101；15,Stenotrophomonas maltophilia K279a；16,
negative control(double-distilled water)；As can be seen that only 1 has occurred LAMP reaction (No. 1 pipe is aobvious
Green is shown, shows to detect the sour klebsiella spp of production), remaining equal color does not change (being still orange), and explanation has
The detection method has high specific.
From the above, it can be seen that calcein/Mn2+Fluorescence developing method is consistent with the result of real-time nephelometry, shows the present invention
The sour klebsiella spp of production LAMP detection method specificity with higher, can specifically detect the primary bar of the sour Cray of production
Bacterium.
Two, the present invention produces the sensitivity of the LAMP detection method of sour klebsiella spp
LAMP detection method of the present invention and the detection of regular-PCR method produce the sensitivity of sour klebsiella spp.
1, LAMP detection method:
Extract produce 700324 total DNA of acid klebsiella spp K.oxytoca ATCC, then with 10 times of gradients (1 times, 10 times,
102Again, 103Again, 104Again, 105Again, 106Being diluted to concentration again) is respectively 48.0ng/ μ l, 4.8ng/ μ l, 480pg/ μ l,
48.0pg/ μ l, 4.8pg/ μ l, 0.48pg/ μ l, 0.048pg/ μ l, then using the DNA through gradient dilution as template, respectively with above-mentioned
One real-time nephelometry and calcein/Mn2+Fluorescence developing method carries out LAMP reaction.
The sour real-time nephelometry LAMP testing result of klebsiella spp is produced as shown in figure 4, code name 1-8 template corresponding concentration is distinguished
Are as follows: 48.0ng/ μ l, 4.8ng/ μ l, 480pg/ μ l, 48.0pg/ μ l, 4.8pg/ μ l, 0.48pg/ μ l, 0.048pg/ μ l, double distillations
Water, it can be seen that the variation of code name 1-5 turbidity generates the sigmoid curve for representing positive reaction, and expression detects the primary bar of the sour Cray of production
Bacterium shows that primer combination of the present invention is 4.8pg/ μ l to the lowest detection sensitivity for producing sour klebsiella spp.
Produce sour klebsiella spp calcein/Mn2+Fluorescence developing method LAMP testing result is as shown in figure 5, code name 1-8 template
Corresponding concentration is respectively as follows: 48.0ng/ μ l, 4.8ng/ μ l, 480pg/ μ l, 48.0pg/ μ l, 4.8pg/ μ l, 0.48pg/ μ l,
0.048pg/ μ l, double distilled water；As can be seen that wherein 1-5 pipe shows green, detects the sour klebsiella spp of production, show
Primer combination of the present invention is also 4.8pg/ μ l to the lowest detection sensitivity for producing sour klebsiella spp.
2, regular-PCR method
Extract produce 700324 total DNA of acid klebsiella spp K.oxytoca ATCC, then with 10 times of gradients (1 times, 10 times,
102Again, 103Again, 104Again, 105Again, 106Being diluted to concentration again) is respectively 48.0ng/ μ l to 0.048pg/ μ l, then through ladder
Spending diluted DNA is template, carries out PCR amplification with the Specific PCR primers of pehX respectively.PCR primer is PCR-F (5 '-
GGATACGGAGTATGCCTTTACG-3 ') and PCR-B
(5 '-GCACCTTGCAGCCCTGA-3 '), amplified fragments are that size is 218bp.
Pcr amplification product electrophoresis detection result as shown in fig. 6, code name 1-8 template corresponding concentration is respectively as follows: 48.0ng/ μ l,
4.8ng/ μ l, 480pg/ μ l, 48.0pg/ μ l, 4.8pg/ μ l, 0.48pg/ μ l, 0.048pg/ μ l, double distilled water, M DNA
marker D2000；As can be seen that target stripe occurs in code name 1-4, show that the detection sensitivity of PCR method is 48.0pg/ μ l.
Compare it is found that the present invention produces the LAMP detection method of sour klebsiella spp, including real-time nephelometry and calcein/
The sample that DNA concentration is 4.8pg/ μ l can be detected in Mn2+ fluorescence developing method, and regular-PCR method is only capable of detecting that DNA is dense
Spend the sample for being 48.0pg/ μ l, and calcein/Mn2+The result of fluorescence developing LAMP method and real-time turbidity LAMP method is consistent,
Show 10 times of high sensitivity of the LAMP detection method of the invention for producing sour klebsiella spp than regular-PCR detection method.
SEQUENCE LISTING
<110>China, PLA Air Force's characteristic medical center disease prevention and control center, the Chinese People's Liberation Army
PLA hospital general
<120>the constant-temperature amplification detection method of the sour klebsiella spp of production and its primer special and kit
<160> 6
<170> PatentIn version 3.5
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cgatctgcgc agcttcac 18
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gtattcagcg tggtgccg 18
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cggacagcgc ggtggttaaa cgaatgtgct ggcttcga 38
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<213>artificial sequence
<400> 5
accgcgcgca ccgtaaa 17
<210> 6
<211> 18
<212> DNA
<213>artificial sequence
<400> 6
gcgcaaaccc gcaaaacg 18
Claims (10)
1. a kind of identification or auxiliary identification produce the ring mediated isothermal amplification primer set of sour klebsiella spp, by primers F 3, primer
B3, primers F IP, primer BIP, primer LoopF and primer LoopB composition；
The primers F 3 is following (a1) or (a2):
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) have by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1 identical
The DNA molecular of function；
The primer B3 is following (a3) or (a4):
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) have by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2 identical
The DNA molecular of function；
The primers F IP is following (a5) or (a6):
(a5) single strand dna shown in the sequence 3 of sequence table；
(a6) have by sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3 identical
The DNA molecular of function；
The primer BIP is following (a7) or (a8):
(a7) single strand dna shown in the sequence 4 of sequence table；
(a8) have by sequence 4 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4 identical
The DNA molecular of function；
The primer LoopF is following (a9) or (a10):
(a9) single strand dna shown in the sequence 5 of sequence table；
(a10) have by sequence 5 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5 identical
The DNA molecular of function；
The primer LoopB is following (a11) or (a12):
(a11) single strand dna shown in the sequence 6 of sequence table；
(a12) have by sequence 6 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5 identical
The DNA molecular of function.
2. primer set according to claim 1, it is characterised in that:
The primers F 3, the primer B3, the primers F IP, the primer BIP, the primer LoopF and the primer
The molar ratio of LoopB is 1:1:8:8:4:4.
3. the ring mediated isothermal amplification reagent that a kind of identification or auxiliary identification produce sour klebsiella spp comprising claims 1 or 2
The primer set.
4. PCR reagent according to claim 3, it is characterised in that: the reagent further include archaeal dna polymerase, dNTP,
Tris-HCl、MgSO4、KCl、(NH4)2SO4, glycine betaine and Tween-20.
5. PCR reagent according to claim 4, it is characterised in that: the reagent further includes fluorescence indicator；
And/or the fluorescence indicator specifically includes calcein and MnCl2。
6. according to the PCR reagent any in claim 3-5, it is characterised in that:
The primers F 3, the primer B3, the primers F IP, the primer BIP, the primer LoopF and the primer
The concentration of LoopB is respectively 0.2 μM, 0.2 μM, 1.6 μM, 1.6 μM, 0.8 μM and 0.8 μM.
7. for detecting the loop-mediated isothermal amplification kit for producing sour klebsiella spp, including it is of any of claims 1 or 2 complete
Any PCR reagent in primer or claim 3-6.
8. in primer set of any of claims 1 or 2 or claim 3-6 any PCR reagent it is following 1) or 2) or
3) application in:
1) preparation identification or auxiliary identification produce the product of sour klebsiella spp；
2) whether contain the product for producing sour klebsiella spp in preparation detection or auxiliary detection sample to be tested；
3) preparation detection or auxiliary detect whether bacterium to be measured is the product for producing sour klebsiella spp.
9. a kind of method that identification or auxiliary identification produce sour klebsiella spp, includes the following steps:
1) genomic DNA of tested microorganism is extracted；
2) genomic DNA extracted using step (1) carries out ring mediated isothermal using primer set described in claim 1 as template
Amplification；
Judged with following A or B:
A, with transmissometer detect amplified production, if amplified production turbidity in S curve change, the tested microorganism be or candidate
To produce sour klebsiella spp；If amplified production turbidity does not change, the tested microorganism is not or candidate is not to produce acid
Klebsiella spp；
B, the fluorescence indicator is added in ring mediated isothermal amplification forward reaction system, observes after reaction,
If amplified production color changes, the tested microorganism is or candidate is to produce sour klebsiella spp；
If amplified production color does not change, the tested microorganism is not or candidate is not to produce sour klebsiella spp.
10. including the following steps: in a kind of detection sample to be tested whether containing the method for producing sour klebsiella spp
(1) total DNA of sample to be tested is extracted；
(2) total DNA extracted using step (1) carries out ring mediated isothermal amplification using primer sets described in claim 1 as template；
Judged with following A or B:
A, amplified production is detected with transmissometer, if the turbid of reaction solution before and after S curve or ring mediated isothermal amplification occurs in amplified production
Degree rises, then the tested microorganism is or candidate is to produce sour klebsiella spp；If amplified production does not occur S curve or ring mediates
The turbidity of reaction solution does not change before and after isothermal duplication, then the tested microorganism is not or candidate is not to produce the primary bar of sour Cray
Bacterium；
B, the fluorescence indicator is added in ring mediated isothermal amplification forward reaction system, observes after reaction, if amplification
Coloured product changes, then the tested microorganism is or candidate is to produce sour klebsiella spp；
If amplified production color does not change, the tested microorganism is not or candidate is not to produce sour klebsiella spp.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628923A (en) * | 2019-10-10 | 2019-12-31 | 中国检验检疫科学研究院 | Primer combination, kit and PSR method for detecting Klebsiella pneumoniae |
| CN114525324A (en) * | 2022-03-31 | 2022-05-24 | 河南冠宇仪器有限公司 | LAMP (loop-mediated isothermal amplification) detection primer group and kit for Burkholderia gladioli and preparation method of freeze-dried microspheres |
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