CN103088139B - Primer pair and standard substance for detecting mycobacteria and application thereof - Google Patents
Primer pair and standard substance for detecting mycobacteria and application thereof Download PDFInfo
- Publication number
- CN103088139B CN103088139B CN201310031783.5A CN201310031783A CN103088139B CN 103088139 B CN103088139 B CN 103088139B CN 201310031783 A CN201310031783 A CN 201310031783A CN 103088139 B CN103088139 B CN 103088139B
- Authority
- CN
- China
- Prior art keywords
- sequence
- mycobacterium
- standard substance
- primer pair
- dna molecule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer pair and a standard substance for detecting mycobacteria and applications thereof. The primer pair is formed by DNA (Deoxyribose Nucleic Acid) respectively shown in a sequence 2 and a sequence 3. The standard substance is a double-stranded DNA molecule shown in the sequence 1 or a plasmid containing the double-stranded DNA molecule shown in the sequence 1 in a sequence table. The special primer pair having the good specificity for the mycobacteria and the standard substance having the generality for mycobacterial strains are provided, so that the special primer pair and the standard substance can be used for the auxiliary identification of the mycobacteria and the quantitative detection of the content of the mycobacteria in a liquid sample solution to be detected or a solid sample solution to be detected by means of fluorescence. The primer pair and the standard substance have the advantages of being high in sensitivity, good in specificity, rapid in detection and the like, and can detect bacteria which cannot be cultured, thereby obtaining a more accurate detection result. Therefore, the technical support is provided for the safety of drinking water and recycled water and the like.
Description
Technical field
The present invention relates to primer pair for detecting mycobacterium and standard substance and their application.
Background technology
Most of mycobacterium is opportunistic pathogen, is often present in natural water, engineering water supply system and indoor water supply and drainage pipe fitting.The mycobacterium of separating from environment is at present more than kind more than 150, and quantity is also being on the increase, and they can cause multi-infection disease in crowd.
At present, culture method is the Gold standard of detection branches bacillus, accurately reliably, but required time longer (4-8 week), and a lot of mycobacterium cannot be cultivated, and current culture method count results only has 1/10th of microscopic counting result.Quantitative fluorescent PCR (qPCR) method refers to and add fluorophor in PCR reaction system, fluorescence is utilized to accumulate the whole PCR process of Real-Time Monitoring, finally by typical curve, unknown template is carried out to the method method of quantitative analysis, have that specificity is stronger, sensitivity is higher, sense cycle is shorter, effectively can solve PCR pollution problem, level of automation advantages of higher.Real-time fluorescence quantitative PCR is adopted quantitatively to confirm this phenomenon to mycobacterium.Adopt the result of qPCR quantitate gene copy number and cell count usually high than culture method about 10 times, consistent with the result of microscope count method.
In addition, the test in laboratory method detected for mycobacterium also comprises gaseous-mass spectrography, liquid phase mass spectrometric analysis.Gaseous mass spectrum and liquid phase mass spectrometric analysis need expensive instrument, are not suitable for conventional sense.
Summary of the invention
The object of this invention is to provide primer pair for detecting mycobacterium and standard substance and their application.
The invention provides the special primer pair for assistant identification mycobacterium, be made up of the Single-stranded DNA fragments shown in the sequence 3 of the Single-stranded DNA fragments shown in the sequence 2 of sequence table and sequence table.
The present invention also protects described special primer to the application in the test kit of preparation assistant identification mycobacterium.
The present invention also protects the test kit of a kind of assistant identification mycobacterium, comprise described special primer to and standard substance; The plasmid of the double chain DNA molecule shown in sequence 1 that described standard substance are sequence table or the double chain DNA molecule shown in sequence 1 containing ordered list.The plasmid of the described double chain DNA molecule shown in sequence 1 containing ordered list specifically can be and is inserted by the double chain DNA molecule shown in the sequence 1 of sequence table
18-T Vector, the recombinant plasmid pMD-18T-hsp65 obtained.
The present invention also protect described special primer to the application of described standard substance in the test kit of preparation assistant identification mycobacterium.
The present invention also protects the application of described primer pair in assistant identification mycobacterium.
The present invention also protects a kind of method of assistant identification mycobacterium; comprise the steps: with the genomic dna of tested bacteria as template; with described special primer to carrying out pcr amplification; if obtain 365 ± 5bp(specifically can be 365bp) pcr amplification product tested bacteria be the mycobacterium of candidate, if do not obtain 365 ± 5bp(specifically can be 365bp) pcr amplification product tested bacteria be the non-branch bacillus of candidate.
The present invention also protect described special primer to or the application of described test kit in detection by quantitative mycobacterium.
The present invention also protect described special primer to or the application of described test kit in the content detecting mycobacterium in water sample to be measured.
The present invention also protects a kind of method of the mycobacterium content detected in water sample to be measured, comprises the steps:
(1) production standard curve as follows; With described standard substance for template, adopt described special primer to carrying out real-time fluorescence quantitative PCR, the Ct value production standard curvilinear equation obtained with the copy number of the standard substance in real-time fluorescence quantitative PCR system and real-time fluorescence quantitative PCR;
(2) from water sample to be measured, extracting genomic dna, is template with genomic dna, adopts described special primer to carrying out real-time fluorescence quantitative PCR, Ct value is substituted into described typical curve equation, obtains the mycobacterium content in water sample to be measured.
In the initial system of described real-time fluorescence quantitative PCR, the concentration of every bar primer that described special primer is right specifically can be 0.2 μm of ol/L.In the response procedures of described real-time fluorescence quantitative PCR, annealing temperature specifically can be 56 ° of C.The response procedures of described real-time fluorescence quantitative PCR specifically can be: (95 ° of C, 5min) × 1 circulation; (95 ° of C, 5s, 56 ° of C annealing 30s, 72 ° of C, 30s) × 40 circulations.
Arbitrary described mycobacterium specifically can be at least one in mycobacterium fortutitum, M. smegmatics, Mycobacterium phlei, Di Shi mycobacterium and yak mycobacterium above.
The invention provides for the good special primer pair of mycobacteria specific, and provide standard substance mycobacterium strain to versatility, can assistant identification mycobacterium, also the mycobacterium content in testing liquid sample or solid sample solution to be measured can be detected by fluorescent quantitation, have highly sensitive, specificity good, detect the advantage such as quick and the bacterium that cannot cultivate can be detected, thus detected result is more accurate.The present invention is that drinking water safety, reuse water safely etc. provide technical support.
Accompanying drawing explanation
Fig. 1 is the result optimizing annealing temperature in real-time quantitative PCR.
Fig. 2 is the result optimizing the primer concentration in initial system in real-time quantitative PCR.
Fig. 3 is the amplification curve in the specificity experiments of embodiment 3.
Fig. 4 is the agarose gel electrophoresis figure in the specificity experiments of embodiment 3.
Fig. 5 is the amplification curve in embodiment 4.
Fig. 6 is the typical curve in embodiment 4.
Fig. 7 is the solubility curve in embodiment 4.
Fig. 8 is the agarose gel electrophoresis figure in embodiment 4.
Fig. 9 is the comparison of the method detection mycobacterium concentration of step 2 and step 3 in embodiment 6.
Figure 10 is the solubility curve in embodiment 6.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
Embodiment 1, special primer are to the preparation with standard substance plasmid
One, the preparation of standard substance plasmid
1, the hsp65 gene order of mycobacteriums all in NCBI is analyzed, based on analytical results, obtain the DNA fragmentation shown in sequence 1 of sequence table.The homology of sequence 1 and the hsp65 gene of each mycobacterium in NCBI is all more than 98%, and do not have homology with other gene, Homoeology comparison result is in table 1.
The sequence analysis result of the hsp65 gene of table 1 sequence 1 and part mycobacterium
2, the double chain DNA molecule shown in sequence 1 of composition sequence table.
3, the double chain DNA molecule of 2-in-1 for step one-tenth is inserted
18-T Vector(TaKaRa Code:D101A), obtain recombinant plasmid pMD-18T-hsp65(standard substance plasmid).
Two, the preparation that special primer is right
A pair special primer is designed as follows based on sequence 1:
The sequence 2 of hsp65-F(sequence table): 5 '-GGAGGACCCGTACGAGAAGA-3 ';
The sequence 3 of hsp65-R(sequence table): 5 '-TGTTGGACTCCTCGACGGTGA-3 '.
Above two primers of synthesis respectively.
The foundation of embodiment 2, mycobacterium quantitative PCR detecting method
Mycobacterium fortutitum (Mycobacteriumfortuitumsubsp.fortuitum): CGMCC is numbered 1.513.
One, the optimization of annealing temperature
1, the genomic dna of mycobacterium fortutitum is extracted.
2, with step 1 extract genomic dna for template, adopt hsp65-F and hsp65-R composition primer pair, adopt
premix ExTaq
tMand by specification carries out quantitative qPCR (Code:DRR041S).
The reaction system (20 μ L) of qPCR:
premix ExTaq
tM10 μ L, hsp65-F, hsp65-R, genomic dna, supply volume to 20 μ L with distilled water.The starting point concentration of hsp65-F and hsp65-R in reaction system is 0.2 μm of ol/L.
The response procedures of qPCR: (95 ° of C, 5min) × 1 circulation; (95 ° of C, 5s, annealing 30s, 72 ° of C, 30s) × 40 circulations; Fluorescence is collected in annealing process; The process of melting curve is: 95 ° of C, 1min, 65 ° of C, 1min, and from 65 ° of C, every 30s temperature raises 0.5 ° of C, and end temp is 95 ° of C, and reaction terminates rear 4 ° of C and preserves.Following annealing temperature is set respectively: 50 ° of C, 51 ° of C, 53 ° of C, 56 ° of C, 58 ° of C, 60 ° of C.
The results are shown in Figure 1.Optimum annealing temperature is 56 ° of C.
Two, the optimization of primer concentration
1, the genomic dna of mycobacterium fortutitum is extracted.
2, with step 1 extract genomic dna for template, adopt hsp65-F and hsp65-R composition primer pair, adopt
premix ExTaq
tMand by specification carries out quantitative qPCR.
The reaction system (20 μ L) of qPCR:
premix ExTaq
tM10 μ L, hsp65-F, hsp65-R, genomic dna, supply volume to 20 μ L with distilled water.The starting point concentration of hsp65-F in reaction system is 0.1 μm of ol/L, 0.2 μm of ol/L, 0.3 μm of ol/L, 0.4 μm of ol/L or 0.5 μm ol/L.The starting point concentration of hsp65-R in reaction system is identical with hsp65-F.
The response procedures of qPCR: (95 ° of C, 5min) × 1 circulation; (95 ° of C, 5s, 56 ° of C annealing 30s, 72 ° of C, 30s) × 40 circulations; Fluorescence is collected in annealing process; The process of melting curve is: 95 ° of C, 1min, 65 ° of C, 1min, and from 65 ° of C, every 30s temperature raises 0.5 ° of C, and end temp is 95 ° of C, and reaction terminates rear 4 ° of C and preserves.
The results are shown in Figure 2.Best primer concentration is 0.2 μm of ol/L.
The specificity experiments that embodiment 3, special primer are right
One, experiment sample
Experiment sample is as follows respectively:
Intestinal bacteria (Escherichia coli): CGMCC is numbered 1.1369.
Salmonella typhimurium (Salmonella typhimurium): CGMCC is numbered 1.1194.
Enterococcus faecalis (Enterococcusfaecalis): CGMCC is numbered 1.2135.
Mycobacterium fortutitum (Mycobacterium fortuitum subsp.fortuitum): CGMCC is numbered 1.513.
M. smegmatics (Mycobacteriumsmegmatis): CGMCC is numbered 1.2621.
Mycobacterium phlei (Mycobacterium phlei): CGMCC is numbered 4.1180.
Di Shi mycobacterium (Mycobacterium diernhoferi): CGMCC is numbered 4.1179.
Yak mycobacterium (Mycobacterium vaccae): CGMCC is numbered 4.1181.
Two, specificity experiments
1, the genomic dna of each experiment sample is extracted.
2, with step 1 extract genomic dna for template, adopt hsp65-F and hsp65-R composition primer pair, adopt
premix ExTaq
tMand by specification carries out quantitative qPCR.
The reaction system (20 μ L) of qPCR:
premix ExTaq
tM10 μ L, hsp65-F, hsp65-R, genomic dna, supply volume to 20 μ L with distilled water.The starting point concentration of hsp65-F and hsp65-R in reaction system is 0.2 μm of ol/L.
The response procedures of qPCR: (95 ° of C, 5min) × 1 circulation; (95 ° of C, 5s, 56 ° of C annealing 30s, 72 ° of C, 30s) × 40 circulations; Fluorescence is collected in annealing process; The process of melting curve is: 95 ° of C, 1min, 65 ° of C, 1min, and from 65 ° of C, every 30s temperature raises 0.5 ° of C, and end temp is 95 ° of C, and reaction terminates rear 4 ° of C and preserves.
Partial results is shown in Fig. 3 (mycobacterium marked in Fig. 3 is mycobacterium fortutitum).Intestinal bacteria, Salmonella typhimurium and enterococcus faecalis all occur without dissolving peak, and mycobacterium fortutitum, M. smegmatics, Mycobacterium phlei, Di Shi mycobacterium and yak mycobacterium all have significantly dissolves peak appearance.Result shows, the special primer that embodiment 1 designs is better to the specificity detecting mycobacterium, with the other kinds bacterium no cross reaction beyond mycobacterium.
3, pcr amplification product is carried out agarose gel electrophoresis, partial results is shown in Fig. 4.In Fig. 4, the corresponding mycobacterium fortutitum of swimming lane 1, the corresponding intestinal bacteria of swimming lane 2, the corresponding Salmonella typhimurium of swimming lane 3, the corresponding enterococcus faecalis of swimming lane 4, the corresponding negative control of swimming lane 5 (water), swimming lane M corresponding DNA Marker(DL2000).Mycobacterium fortutitum, M. smegmatics, Mycobacterium phlei, Di Shi mycobacterium and yak mycobacterium all show the fragment of about 356bp, and other bacterium does not all show and obtains pcr amplified fragment.Result shows, fragment for the purpose of the fragment of quantitative pcr amplification, the other kinds bacterium no cross reaction beyond the special primer pair that embodiment 1 designs and mycobacterium.
Between the detectability of embodiment 4, quantitative PCR detecting method, quantification area, typical curve and specificity
With recombinant plasmid pMD-18T-hsp65 for template, adopt the primer pair of hsp65-F and hsp65-R composition, adopt
premix ExTaq
tMand by specification carries out qPCR (Code:DRR041S).
The reaction system (20 μ L) of qPCR: 2 ×
premix ExTaq
tM10 μ L, hsp65-F, hsp65-R, 2 μ L recombinant plasmid pMD-18T-hsp65 solution, supply volume to 20 μ L with distilled water.The starting point concentration of hsp65-F and hsp65-R in reaction system is 0.2 μm of ol/L.Recombinant plasmid pMD-18T-hsp65 solution is that TE damping fluid recombinant plasmid pMD-18T-hsp65 being dissolved in pH8.0 obtains.In recombinant plasmid pMD-18T-hsp65 solution, the concentration of recombinant plasmid pMD-18T-hsp65 is 1.8 × 10
2, 1.8 × 10
3, 1.8 × 10
4, 1.8 × 10
5, 1.8 × 10
6, 1.8 × 10
7, 1.8 × 10
8, 1.8 × 10
9or 1.8 × 10
10individual copy/μ L.
The response procedures of qPCR: (95 ° of C, 5min) × 1 circulation; (95 ° of C, 5s, 56 ° of C, 30s, 72 ° of C, 30s) × 40 circulations; Fluorescence is collected in annealing process; The process of melting curve is: 95 ° of C, 1min, 65 ° of C, 1min, and from 65 ° of C, every 30s temperature raises 0.5 ° of C, and end temp is 95 ° of C, and reaction terminates rear 4 ° of C and preserves.
Amplification curve is shown in Fig. 5.As can be seen from the figure, amplification curve is level and smooth, and expanding effect is better.
With the copy number denary logarithm of recombinant plasmid pMD-18T-hsp65 in each PCR system for X-coordinate, set up the typical curve of real-time quantitative PCR with critical cycle number (Threshold Cycle, Ct) for ordinate zou, see Fig. 6, typical curve equation is Y=-3.2831x+38.862, R
2=0.9937.Lowest detection is limited to 3.6 × 10
2individual copy/PCR system, detection by quantitative interval is 3.6 × 10
2-3.6 × 10
10individual copy/PCR system, amplification efficiency E=10
1/3.2831-1=1.016, namely 101.6%.
These results suggest that, adopt the special primer pair that embodiment 1 designs, detected the standard substance plasmid of embodiment 1 preparation by qPCR, efficiency is high, and linear relationship is good, meets the requirement preparing real-time quantitative PCR typical curve.
Whether the melting point curve of real-time quantitative PCR is mainly specificity object product to detect PCR primer.The based composition of DNA double chain is different, and melting temperature is different.When arriving the melting temperature of product and causing DNA double chain to be opened, discharge with the SYBR Green fluorescence dye of DNA double chain combination and cause fluorescent value and reduce.If reaction system and condition optimizing obtain very well, the specificity of primer is very high, and PCR primer purity is high, then the fusing point peak of melting point curve is narrow and sharp, if product is impure, have nonspecific reaction product and primer dimer, then there is several peak at fusing point peak.Solubility curve is shown in Fig. 7, and curve is steady, and peak is sharp and narrow, and melting temperature (Tm) is 93 ± 1 ° of C, proves that this pcr amplification product is very special.
Real-time quantitative PCR product is carried out agarose electrophoresis, and result as shown in Figure 8.In Fig. 8, swimming lane M corresponding DNA Marker(DL2000), the corresponding negative control (water) of swimming lane N, swimming lane 1-9 is corresponding in turn to that to add concentration be 1.8 × 10
2, 1.8 × 10
3, 1.8 × 10
4, 1.8 × 10
5, 1.8 × 10
6, 1.8 × 10
7, 1.8 × 10
8, 1.8 × 10
9or 1.8 × 10
10the PCR system of the recombinant plasmid pMD-18T-hsp65 solution of individual copy/μ L, swimming lane 10 is positive control (mycobacterium fortutitum).As seen from the figure, pcr amplification product length is about 356bp, and this qPCR atopic is strong.
The repeatability of embodiment 5, quantitative PCR detecting method
Recombinant plasmid pMD-18T-hsp65 is prepared continuous 7 times according to the method for embodiment 1, obtain the recombinant plasmid pMD-18T-hsp65 of 7 batches, by recombinant plasmid pMD-18T-hsp65 multigelation 4 times, carry out qPCR mensuration, the variation coefficient (CV) according to cycle number is evaluated repeatability.The results are shown in Table 2.
Between table 2pMD-18T-hsp65 different batches, repeatability is analyzed
Result is as shown in table 2, and the cycle number variation coefficient of plasmid standard pMD-18T-hsp65 is respectively 1.39%-7.58%, and these results show, the typical curve repeatability of standard substance plasmid pMD-18T-hsp65 is good.
Embodiment 6, quantifying PCR method is utilized to detect mycobacterium in actual environmental water sample
One, actual water sample collection
Actual water sample is taken from Qinghe, Beijing regeneration water factory two and to be settled out water, film water outlet, and sample time is in December ,-2012 in October, 2012, gets 3 times altogether, gathers 5L water sample at every turn.The sample fetched is put into ice chest and is transported laboratory back, 4 DEG C of preservations.
Two, the mycobacterium in culture method detection actual sample is adopted
The mycobacterium detected in actual sample adopts filter membrane method to measure, and uses Middlebrook7H10 substratum (Difco
tMmiddlebrook7H10Agar) and corresponding nutritional additive (Middlebrook OADCEnrichment), adopt bacterial inhibitor cetylpyridinium chloride simultaneously.First by water sample according to 10 times of gradient dilutions, cetylpyridinium chloride (it is the cetylpyridinium chloride of 0.005% that every 100mL water sample adds 500 μ L mass percentage) is added in water sample after dilution, then the sample of each gradient dilution of membrane filtration 100mL of 0.45 μm is adopted, then filter membrane is laid on substratum, notes there is no bubble between filter membrane and substratum.Be inverted by substratum, cultivate 7 days at 35 ± 2 DEG C, on each substratum, Stochastic choice 3 mono-clonals, confirm by Ziehi-Neelsen stain.
Three, the mycobacterium in quantitative PCR detection actual water sample is adopted
1, water sample concentrates
Because in reuse water, the concentration of mycobacterium is lower, collect the mycobacterium in sample by filter membrane method.
(1) adopt the membrane filtration 1L water sample of 0.45 μm, then taken off by filter membrane aseptic nipper, be placed in the 5mL centrifuge tube that 4mL sterile PBS buffer is housed, whirlpool shakes, and is moved on to by liquid rotating in 10mL centrifuge tube.
(2) in the 5mL centrifuge tube of completing steps (1), the aseptic PBS damping fluid of 4mL is added again, whirlpool shakes, liquid is also transferred in the 10mL centrifuge tube that step (1) obtains, 4 DEG C, the centrifugal 15min of 12000rpm, remove 6mL supernatant liquor, the mixing of the material pipettor of remainder is collected in 2mL centrifuge tube, 4 DEG C, the centrifugal 5min of 12000rpm, remove supernatant liquor, retain remaining sample and be about 100-200 μ L.
2, the mycobacterium in quantitative PCR detection actual water sample is adopted
(1) genomic dna of the product providing step 1 to obtain.
(2) take genomic dna as template, adopt the primer pair of hsp65-F and hsp65-R composition, adopt
premix ExTaq
tMand by specification carries out qPCR (Code:DRR041S).
The reaction system (20 μ L) of qPCR:
premix ExTaq
tM10 μ L, hsp65-F, hsp65-R, 9 μ L genomic dnas, supply volume to 20 μ L with distilled water.The starting point concentration of hsp65-F and hsp65-R in reaction system is 0.2 μm of ol/L.
The response procedures of qPCR: (95 ° of C, 5min) × 1 circulation; (95 ° of C, 5s, 56 ° of C, 30s, 72 ° of C, 30s) × 40 circulations; Fluorescence is collected in annealing process; The process of melting curve is: 95 ° of C, 1min, 65 ° of C, 1min, and from 65 ° of C, every 30s temperature raises 0.5 ° of C, and end temp is 95 ° of C, and reaction terminates rear 4 ° of C and preserves.
The result of quantitative PCR is substituted into the typical curve equation that embodiment 4 obtains, obtain the copy number of goal gene in water sample to be measured, the copy number of a goal gene represents a mycobacterium.
What the method (culture method) of step 2 and the method (qPCR method) of step 3 detected mycobacterium concentration in water sample the results are shown in Table 3.
Table 3 utilizes culture method and qPCR to detect the concentration of mycobacterium in water sample
Method in order to comparison step two and step 3 detects the consistence of mycobacterium concentration, and the concentration mapping of the mycobacterium two kinds of methods detected, as shown in Figure 9, X-coordinate is the concentration (Log of the mycobacterium in the water sample to be measured utilizing culture method to count to result
10cFU/100mL), ordinate zou is the concentration (Log utilizing qPCR method to detect the mycobacterium hsp65 gene in water sample to be measured
10copy number/100mL), the result linear dependence of two kinds of detection methods is better, (Y=0.3717X+3.9957), the good (R of dependency
2=0.7211), illustrate invention provide quantifying PCR method accurately credible.In addition, comparatively culture method is higher for qPCR detected result.
In the method for step 3, the melting curve of qPCR is shown in Figure 10, and curve is steady, and the fusing point peak of melting point curve is narrow and sharp, and melting temperature (Tm) is 93 ± 1 ° of C, proves that pcr amplification product is very special.
The above results shows that the result of the detection of real-time quantitative PCR provided by the invention is comparatively reliable, and primer pair specificity is high, also shows that plasmid standard prepared in the present invention has good linear relationship and sensing range.
Claims (8)
1., for the primer pair of assistant identification mycobacterium, be made up of the Single-stranded DNA fragments shown in the sequence 3 of the Single-stranded DNA fragments shown in the sequence 2 of sequence table and sequence table.
2. the application of primer pair described in claim 1 in the test kit of preparation assistant identification mycobacterium.
3. a test kit for assistant identification mycobacterium, comprises primer pair described in claim 1 and standard substance; The plasmid of the double chain DNA molecule shown in sequence 1 that described standard substance are sequence table or the double chain DNA molecule shown in sequence 1 containing ordered list.
4. test kit as claimed in claim 3, is characterized in that: the plasmid of the described double chain DNA molecule shown in sequence 1 containing ordered list is for inserting the double chain DNA molecule shown in the sequence 1 of sequence table
the recombinant plasmid pMD-18T-hsp65 obtained.
5. primer pair described in claim 1 and the standard substance application in the test kit of preparation assistant identification mycobacterium; The plasmid of the double chain DNA molecule shown in sequence 1 that described standard substance are sequence table or the double chain DNA molecule shown in sequence 1 containing ordered list.
6. apply as claimed in claim 5, it is characterized in that: the plasmid of the described double chain DNA molecule shown in sequence 1 containing ordered list is for inserting the double chain DNA molecule shown in the sequence 1 of sequence table
the recombinant plasmid pMD-18T-hsp65 obtained.
7. the application of test kit described in primer pair described in claim 1 or claim 3 in the content detecting mycobacterium in water sample to be measured.
8. detect a method for the mycobacterium content in water sample to be measured, comprise the steps:
(1) production standard curve as follows; With described standard substance for template, adopt special primer described in claim 1 to carrying out real-time fluorescence quantitative PCR, the Ct value production standard curvilinear equation obtained with the copy number of the standard substance in real-time fluorescence quantitative PCR system and real-time fluorescence quantitative PCR; The plasmid of the double chain DNA molecule shown in sequence 1 that described standard substance are sequence table or the double chain DNA molecule shown in sequence 1 containing ordered list;
(2) from water sample to be measured, extracting genomic dna, take genomic dna as template, adopting special primer described in claim 1 to carrying out real-time fluorescence quantitative PCR, Ct value being substituted into described typical curve equation, obtains the mycobacterium content in water sample to be measured.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310031783.5A CN103088139B (en) | 2013-01-28 | 2013-01-28 | Primer pair and standard substance for detecting mycobacteria and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310031783.5A CN103088139B (en) | 2013-01-28 | 2013-01-28 | Primer pair and standard substance for detecting mycobacteria and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103088139A CN103088139A (en) | 2013-05-08 |
| CN103088139B true CN103088139B (en) | 2015-04-22 |
Family
ID=48201266
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310031783.5A Expired - Fee Related CN103088139B (en) | 2013-01-28 | 2013-01-28 | Primer pair and standard substance for detecting mycobacteria and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103088139B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628619B (en) * | 2019-01-03 | 2022-05-03 | 首都医科大学附属北京胸科医院 | SNP molecular marker and method for identifying mycobacteria, primer composition, kit and application |
| CN113718019A (en) * | 2021-09-03 | 2021-11-30 | 上海城市水资源开发利用国家工程中心有限公司 | Method for evaluating biological safety of water supply system by using sodB gene |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532053A (en) * | 2008-12-19 | 2009-09-16 | 上海天昊生物科技有限公司 | Method for identifying mycobacterium |
| CN102108398A (en) * | 2010-12-01 | 2011-06-29 | 广东省中医院 | Fluorescent quantitative PCR detection method for Mycobacterium tuberculosis |
| CN102433384A (en) * | 2011-12-19 | 2012-05-02 | 广东出入境检验检疫局检验检疫技术中心 | Primer and polymerase chain reaction-denatured high performance liquid chromatography (PCR-DHPLC) kit for detecting mycobacteria |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827944B (en) * | 2012-09-19 | 2014-07-23 | 广州华峰生物科技有限公司 | Mycobacterium detection kit and using method thereof |
-
2013
- 2013-01-28 CN CN201310031783.5A patent/CN103088139B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532053A (en) * | 2008-12-19 | 2009-09-16 | 上海天昊生物科技有限公司 | Method for identifying mycobacterium |
| CN102108398A (en) * | 2010-12-01 | 2011-06-29 | 广东省中医院 | Fluorescent quantitative PCR detection method for Mycobacterium tuberculosis |
| CN102433384A (en) * | 2011-12-19 | 2012-05-02 | 广东出入境检验检疫局检验检疫技术中心 | Primer and polymerase chain reaction-denatured high performance liquid chromatography (PCR-DHPLC) kit for detecting mycobacteria |
Non-Patent Citations (1)
| Title |
|---|
| 谭笑.基于hsp65基因的PCR技术在检测临床标本中结核杆菌的应用.《湖南师范大学学报(医学版)》.2012,第9卷(第4期),第61-64页,尤其第1.3节PCR扩增. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103088139A (en) | 2013-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Song et al. | Combining tag-specific primer extension and magneto-DNA system for Cas14a-based universal bacterial diagnostic platform | |
| Xiang et al. | High-throughput microfluidic strategy based on RAA-CRISPR/Cas13a dual signal amplification for accurate identification of pathogenic Listeria | |
| Weiss et al. | An extended multilocus sequence typing (MLST) scheme for rapid direct typing of Leptospira from clinical samples | |
| Parra et al. | Development of a molecular diagnostic test applied to experimental abattoir surveillance on bovine tuberculosis | |
| Wu et al. | CRISPR-Cas system meets DNA barcoding: Development of a universal nucleic acid test for food authentication | |
| Tone et al. | Enhancing melting curve analysis for the discrimination of loop-mediated isothermal amplification products from four pathogenic molds: Use of inorganic pyrophosphatase and its effect in reducing the variance in melting temperature values | |
| Christensen et al. | Real-time PCR for universal phytoplasma detection and quantification | |
| Straub et al. | Estimated copy number of Bacillus anthracis plasmids pXO1 and pXO2 using digital PCR | |
| CN103276057B (en) | LAMP technology based rapid Botrytis cinerea detection method | |
| Mehle et al. | A real-time PCR detection system for the bois noir and flavescence dorée phytoplasmas and quantification of the target DNA | |
| CN103088139B (en) | Primer pair and standard substance for detecting mycobacteria and application thereof | |
| CN105907861A (en) | Method and primer for quick detection and classification of mycobacteria | |
| CN103993090B (en) | To Providence O31, O41, O42, the nucleotides that O43 and O50 are special and application thereof | |
| Kang et al. | Comparison of DNA extraction methods for drug susceptibility testing by allele-specific primer extension on a microsphere-based platform: Chelex-100 (in-house and commercialized) and MagPurix TB DNA Extraction Kit | |
| CN105256052B (en) | A kind of kit and its detection method for Legionella quick detection and parting | |
| CN102277439B (en) | Rapid detection kit for goat contagious pleuropneumonia and preparation method thereof | |
| Di Marco | Real-time PCR detection of Mycoplasma pneumoniae in the diagnosis of community-acquired pneumonia | |
| CN102533981B (en) | Primer and detection method as well as kit for rapidly detecting bacillus subtilis | |
| Aviv et al. | Real-time reverse transcription PCR as a tool to study virulence gene regulation in bacterial pathogens | |
| CN105154538A (en) | Primer and method for rapidly detecting and parting legionella | |
| CN102732601B (en) | Kit for diagnosis of tuberculosis based on isothermal nucleic acid amplification technique | |
| JP2008536518A5 (en) | ||
| CN110747261A (en) | Specific primer, detection method and application of tetracycline antibiotic resistance gene tetX | |
| Toro et al. | Bacterial group II introns: identification and mobility assay | |
| Schembri et al. | Multi-target plasmid controls for conventional and real-time PCR-based serotyping of Streptococcus pneumoniae |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150422 Termination date: 20180128 |