CN102367483B - Specific primer pair for assisting identification of Klebsiella pneumoniae - Google Patents
Specific primer pair for assisting identification of Klebsiella pneumoniae Download PDFInfo
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- CN102367483B CN102367483B CN 201110349331 CN201110349331A CN102367483B CN 102367483 B CN102367483 B CN 102367483B CN 201110349331 CN201110349331 CN 201110349331 CN 201110349331 A CN201110349331 A CN 201110349331A CN 102367483 B CN102367483 B CN 102367483B
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Abstract
The invention discloses a specific primer pair for assisting identification of Klebsiella pneumoniae. The primer pair provided in the invention consists of DNA shown in sequence 1 of the sequence table and DNA shown in sequence 2 of the sequence table. The specific primer pair designed in the invention can be used for assisting identification about whether Bactrocera dorsalis (Hendel) carries Klebsiella pneumoniae only by means of PCR (polymerase chain reaction) and electrophoresis, and the identification is characterized by high sensitivity, simple process and no need for cloning. In order to increase accuracy, a PCR amplified product can be subjected to sequencing. The specific primer pair provided in the invention improves the efficiency of Bactrocera dorsalis (Hendel) symbiotic bacteria (Klebsiella pneumoniae), and creates conditions for further study of the coevolution relation between Bactrocera dorsalis (Hendel) and Klebsiella pneumoniae.
Description
Technical field
The present invention relates to a kind of special primer pair of assistant identification Klebsiella pneumonia.
Background technology
Citrus fruit fly Bactrocera (Bactrocera) dorsalis (Hendel) has another name called orient fruit fly, is under the jurisdiction of Diptera (Diptera), Tephritidae (Tephritidae), Bactrocera (Bactrocera).This worm host range is wide, can endanger 250 various fruits of section more than 40 and the vegetables such as oranges and tangerines, piscidia, carambola, is a kind of worldwide quarantine pest insect.Citrus fruit fly is from intruding into China abroad, and at home from reaching the north diffusion in the south, and the trend of further diffusion is increasingly severe, to the export mixes of the main fruit of China larger threat.At present, citrus fruit fly mainly is distributed in the provinces and regions such as Guangdong, Guangxi, Hunan, Guizhou, Fujian, Hainan, Yunnan, Sichuan, Taiwan.Distribute widely, and the serious harm that China's agricultural and forestry production and agroecosystem are caused, so that the method for preventing and treating of citrus fruit fly and invasion mechanism become problem in the urgent need to address.
The trypetid fungal component be mostly can depolymerized pectin bacterium and vinelandii, help Host Digestion food and carry out the carbon nitrogen cycle; And final by affecting mating, reproduction and life-span, affect trypetid to the adaptive faculty of environment.In addition, the fungal component that is separated in the middle of the trypetid can also produce volatile matter, and the host is had certain attracting ability, can also be as host's food.Therefore, research citrus fruit fly endosymbiosis bacterium with and with the cooperative intrusion effect of host trypetid, for the invasion approach that cuts off trypetid, find new trypetid bait, improve the adaptive capacity to environment of sterile trypetid, further realize " antibiotic insect protected ", and it is significant to find that new trypetid prevents and treats method.
The research method of tradition trypetid fungal component comprises for the separation and Culture authenticate technology that can cultivate bacterium, for the Protocols in Molecular Biology that can not cultivate bacterium and micro-imaging technique.
Protocols in Molecular Biology grows up for cultivating fungal component, mainly comprises structure and denaturing gradient gel electrophoresis (PCR-DGGE) technology of fungal component 16s rRNA gene library.The PCR-DGGE technology is extracted first grand genome in the trypetid body, and universal primer amplification fungal component 16s rRNA gene fragment then by the DGGE electrophoretic technique with not homotactic gene fragment separately, checks order, and determines the kind of endosymbiosis bacterium by sequential analysis.The method quicklook; Be convenient to the comparison between Different Individual or the sample, be conducive to find its identical or different fungal component.But because the defective of electrophoresis itself, the method can only be separated the following fragment of 500bp, and the sequence information of acquisition is comprehensive not; And fungal component of the same race may have different electrophoretic bands; Affected the accuracy of the method; In addition, this technological operation is loaded down with trivial details, and operator's state of the art is had relatively high expectations.By contrast, structure 16s rRNA gene library can obtain the details about fungal component 16s rDNA sequence, is conducive to the citrus fruit fly fungal component is cooked a detailed examination.
The method that adopt to make up 16s rRNA gene library is screened citrus fruit fly endosymbiosis bacterium kind, the general universal primer (27F/1492R) that adopts for 16s rDNA sequence, can amplify germy length be about the 16srDNA sequence of 1.5kb.Because comprising the 16s rDNA sequence of multiple fungal component, show bimodally during PCR product direct Sequencing, can't obtain preferably experimental result.Need further sequence to be reclaimed, be connected on the carrier, transform intestinal bacteria, select the positive colony that inserts the purpose fragment and check order.In this process, need select a large amount of clones and carry out bacterium colony PCR and order-checking, just can detect all fungal components, the method very complicated, sensitivity is not high, very easily ignores the lower fungal component of certain class content.Therefore, improve the fungal component detection sensitivity, optimize the key that the detection method of certain fungal component is become the research of citrus fruit fly endosymbiosis bacterium.
Summary of the invention
The special primer pair that the purpose of this invention is to provide a kind of assistant identification Klebsiella pneumonia.
The special primer of assistant identification Klebsiella pneumonia provided by the invention (Klebsiella pneumoniae) pair forms (downstream primer) by DNA shown in the sequence 2 of DNA (upstream primer) shown in the sequence 1 of sequence table and sequence table.
Described special primer is to can be used for the assistant identification Klebsiella pneumonia.
Described special primer is to can be used for preparing the test kit of assistant identification Klebsiella pneumonia.
The present invention also protects a kind of test kit of assistant identification Klebsiella pneumonia, comprises described special primer pair.
The present invention also protects a kind of method of assistant identification Klebsiella pneumonia; comprise the steps: that genomic dna (or diluent of described genomic dna) take bacterium to be measured (eubacterium or claim bacterium) is as template; with described special primer to carrying out pcr amplification; be candidate's Klebsiella pneumonia if obtain pcr amplification product bacterium to be measured, be not candidate's non-Klebsiella pneumonia if obtain pcr amplification product bacterium to be measured.
The size of described pcr amplification product can be 500bp-750bp, specifically can be 500bp-700bp, more specifically can be 558bp-565bp, is preferably 558bp.
The reaction system of described pcr amplification (50 μ L) specific as follows: 5 μ L, 10 * Reaction Buffer (contains Mg
2+), 4 μ L dNTP mixture (2.5mM), Taq enzyme 0.5 μ L (2.5U/ μ L), ddH
2O 37.5 μ L, described template 1 μ L, described upstream primer (10 μ M) 1 μ L, described downstream primer (10 μ M) 1 μ L.
The reaction parameter of described pcr amplification specifically can be: 94 ℃ of 3min; 94 ℃ of 30sec, 59.6 ℃ of 30sec, 72 ℃ of 30sec, 35 circulations; 72 ℃ of 5min.
Klebsiella pneumonia is a kind of in the citrus fruit fly endosymbiosis bacterium.
The present invention also protects described special primer to whether carry the application in the Klebsiella pneumonia at the assistant identification citrus fruit fly.
Described special primer is to can be used for preparing the test kit whether the assistant identification citrus fruit fly carries Klebsiella pneumonia.
The present invention also protects a kind of assistant identification citrus fruit fly whether to carry the test kit of Klebsiella pneumonia, comprises described special primer pair.
The present invention also protects a kind of assistant identification citrus fruit fly whether to carry the method for Klebsiella pneumonia; comprise the steps: that genomic dna take citrus fruit fly to be measured is as template; with described special primer to carrying out pcr amplification; carry Klebsiella pneumonia if obtain the described citrus fruit fly of pcr amplification product, do not carry Klebsiella pneumonia if obtain the described citrus fruit fly of pcr amplification product.
The size of described pcr amplification product can be 500bp-750bp, specifically can be 500bp-700bp, more specifically can be 558bp-565bp, is preferably 558bp.
The reaction system of described pcr amplification (50 μ L) specific as follows: 5 μ L, 10 * Reaction Buffer (contains Mg
2+), 4 μ L dNTP mixture (2.5mM), Taq enzyme 0.5 μ L (2.5U/ μ L), ddH
2O 37.5 μ L, described template 1 μ L, described upstream primer (10 μ M) 1 μ L, described downstream primer (10 μ M) 1 μ L.
The reaction parameter of described pcr amplification specifically can be: 94 ℃ of 3min; 94 ℃ of 30sec, 59.6 ℃ of 30sec, 72 ℃ of 30sec, 35 circulations; 72 ℃ of 5min.
The a pair of special primer that the present invention is directed in the citrus fruit fly different geographic populations ubiquitous Klebsiella pneumonia 16s rDNA sequences Design can be directly used in and detect in the citrus fruit fly sample whether have Klebsiella pneumonia.Whether the special primer of using the present invention's design to only can carrying Klebsiella pneumonia by the assistant identification citrus fruit fly by PCR and electrophoresis, highly sensitive, step simple, need not the clone, in order to increase accuracy, pcr amplification product can also be checked order.The present invention has improved the efficient that citrus fruit fly fungal component (Klebsiella pneumonia) detects, for the coevolution relation of further studying citrus fruit fly and Klebsiella pneumonia has been created condition.
Description of drawings
Fig. 1 is that the PCR among the embodiment 2 identifies electrophorogram.
Fig. 2 is that the PCR among the embodiment 3 identifies electrophorogram.
Fig. 3 is that the PCR among the embodiment 5 identifies electrophorogram.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
Klebsiella pneumonia (Klebsiella pneumoniae): belong to γ-distortion Gammaproteobacteria (Gammaproteobacteria), enterobacteria order (Enterobacteriales), enterobacteriaceae (Enterobacteriaceae), Klebsiella (Klebsiella) is the type species of Klebsiella; The public can obtain from China Agricultural University; The reference of mentioning Klebsiella pneumonia is: Wang Jing, HAN LIXIA, Zhang Fenghua, Li Jian.In the groove the application in detecting Klebsiella Pneumoniae institute of repeat sequence primer polymerase chain reaction.The great variety of modern medical examining magazine.2010，25(5)：28-30.。
The citrus fruit fly sample that is used for embodiment 1 and embodiment 3 has following several: sample 1: the female worm of citrus fruit fly that Inspection and Quarantine Technic Center, Guangdong Entry-Exit Inspection and Qu raises; Sample 2: gather the citrus fruit fly male worm from Thailand; Sample 3: gather the citrus fruit fly male worm from Shanghai Baoshan; Sample 4: gather the citrus fruit fly male worm from Qingpu Shanghai; Sample 5: gather the citrus fruit fly male worm from Bay, Ningde, Fujian Province; Sample 6: gather the citrus fruit fly male worm from Foochow, Fujian; Sample 7: gather the citrus fruit fly male worm from the Qionghai; Sample 8: gather the citrus fruit fly male worm from Shanghai International Automobile City Tourist Festival; Sample 9: gather the citrus fruit fly male worm from Songjiang, Shanghai; Sample 10: gather the citrus fruit fly male worm from Ningbo of Zhejiang; Sample 11: gather the citrus fruit fly male worm from Xiamen of Fujian Province; Sample 12: gather the citrus fruit fly male worm from Guangzhou Guangdong; Sample 13: gather the citrus fruit fly male worm from the Pingxiang, Guangxi; Sample 14: gather the citrus fruit fly male worm from Jinghong In Yunnan Province; Sample 15: gather the citrus fruit fly male worm from Xingyi of Guizhou; Sample 16: gather the citrus fruit fly male worm from the Nanning; Sample 17: the citrus fruit fly male worm that Inspection and Quarantine Technic Center, Guangdong Entry-Exit Inspection and Qu raises.The public can obtain the citrus fruit fly sample from China Agricultural University, and the reference of citrus fruit fly is as follows: careful, and Wang Yingbo, Xiao Tieguang, Zhou Shewen.Citrus fruit fly biology and study on prevention.Crop investigations.2009，23(S1)：84-86.。
One, the acquisition of Klebsiella pneumonia 16s rDNA sequence
Respectively each citrus fruit fly sample (sample 1 is to sample 17) is carried out following evaluation:
1, the extraction of citrus fruit fly genomic dna
The blood of employing sky, Beijing root biochemical technology company limited/cell/tissue genome DNA extracting reagent kit extracts the genomic dna of the whole polypide of single head adult.
2, adopt the universal primer of 16s rDNA sequence to carry out pcr amplification
Utilize the 16s rDNA sequence of the universal primer 27F (5 '-GAGAGTTTGATCCTGGCTCAG-3 ') of bacterium 16s rDNA sequence and 1492R (5 '-TACGGTTACCTTGTTACGACTT-3 ') amplification citrus fruit fly symbiotic bacterium, target amplification product length is about 1500bp.
The pcr amplification cumulative volume is 50 μ L, and wherein 5 μ L, 10 * Reaction Buffer (contains Mg
2+), 4 μ L dNTPmixture (2.5mM), Taq enzyme 0.5 μ L (2.5U/ μ L, sky, Beijing root biochemical technology company limited), ddH
2O37.5 μ L, template 1 μ L (115.8ng/ μ L), 27F (10 μ M) 1 μ L, 1492R (10 μ M) 1 μ L.
The pcr amplification condition is: the thermal cycling program is 95 ℃ of 5min, then carries out 35 circulations (94 ℃ of 3min, 55 ℃ of 30s, 72 ℃ of 1min), finishes behind 72 ℃ of reaction 10min at last.
Amplified production is the mixture of all eubacterial 16s rDNA fragments.
3, the structure in citrus fruit fly fungal component 16s rDNA library
Adopt agarose gel electrophoresis to detect the quality of PCR product, and adopt the gel of sky, Beijing root biochemical technology company limited to reclaim test kit recovery product.Be connected on the PMD19-T carrier of precious biotech firm reclaiming product, 16 ℃ of connections are spent the night, and transform bacillus coli DH 5 alpha (available from the Beijing Quanshijin Biotechnology Co., Ltd), carry out blue hickie screening at ammonia benzyl resistance LB culture medium flat plate.Employing carrier universal primer RV-M (5 '-GAGCG-GATAACAATTTCACACAGG-3 ') and M13-47 (5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ') (bacterium colony PCR reaction conditions is: 95 ℃ of sex change 5min to carry out bacterium colony PCR detection positive colony; 94 ℃ of sex change 1min, 58 ℃ of renaturation 1min, 72 ℃ are extended 2min, 31 circulations; 72 ℃ are fully extended 10min), amplified production adopts 1.5% agarose gel electrophoresis detection, and selecting wherein, the clone of clip size correct (having inserted amplified fragments) checks order.Send the two-way order-checking of company (the prosperous biotechnology of Beijing AudioCodes limited liability company) with positive colony, above-mentioned carrier universal primer is adopted in order-checking.Each library is selected at least 60 positive colonies and is checked order, and finishes the structure in citrus fruit fly symbiotic bacterium 16s rDNA library.
4, the acquisition of Klebsiella pneumonia 16s rDNA sequence
The sequence of survey time in the library is committed to NCBI compares online the kind of clear and definite citrus fruit fly endosymbiosis bacterium.The library of the endosymbiosis bacterium by the citrus fruit fly that makes up, the citrus fruit fly fungal component comprises 16 kinds altogether, wherein a kind of is Klebsiella pneumonia.
Sequencing result shows, sample 1 has the conserved sequence (be judged as and contain Klebsiella pneumonia) of Klebsiella pneumonia to the sample 9, and sample 10 does not have the conserved sequence (be judged as and do not contain Klebsiella pneumonia) of Klebsiella pneumonia to the sample 17.
Two, for the right design of special primer of Klebsiella pneumonia 16s rDNA sequence
1, the right design of special primer
After the fungal component 16s rDNA sequence of 16 kinds of citrus fruit fly of obtaining carried out the DNAman compare of analysis, design was for the special primer of Klebsiella pneumonia pair, and sequence is as follows:
Upstream primer (sequence 1 of sequence table): 5 '-GTGAGGTTAATAACCTCATCGATTGACG-3 ';
Downstream primer (sequence 2 of sequence table): 5 '-CTGGAAAGTTCTGGTGGATGTCAAGACC-3 '.
2, the right design of special primer
Adopt Oligo 6.0 softwares that the primer pair indices is assessed, assessment content and evaluation criteria are as follows: the Delta G value absolute value of upstream and downstream primer is no more than 9; Can form primer dimer or hairpin structure in conjunction with base pair not above 3; GC% content is controlled between the 30%-70%, and does not differ too large between the upstream and downstream; The mistake efficiency of initiation is no more than 100; At last, to provide the most suitable annealing temperature of this primer pair be 59.6 ℃ to Oligo 6.0 softwares.
1, adopt bacterial genomes DNA extraction test kit (sky, Beijing root biochemical technology company limited) to extract the genomic dna of Klebsiella pneumonia.
2, the genomic dna that extracts with step 1 (take water as negative control) is as template, and the special primer that designs with the step 2 of embodiment 1 obtains pcr amplification product to carrying out pcr amplification.
Pcr amplification volume (50 μ L): 5 μ L, 10 * Reaction Buffer (contains Mg
2+), 4 μ L dNTP mixture (2.5mM), Taq enzyme 0.5 μ L (2.5U/ μ L, sky, Beijing root biochemical technology company limited), ddH
2O 37.5 μ L, genomic dna 1 μ L (115.8ng/ μ L), upstream primer (10 μ M) 1 μ L, downstream primer (10 μ M) 1 μ L.
Pcr amplification condition: 94 ℃ of 3min; 94 ℃ of 30sec, 59.6 ℃ of 30sec, 72 ℃ of 30sec, 35 circulations; 72 ℃ of 5min.
3, get 5 μ L pcr amplification products, carry out 1.5% agarose gel electrophoresis and ethidium bromide (EB) dyeing, in gel imaging system, observe and imaging (Gel Logical Pro 212).The results are shown in Figure 1, between 500bp-750bp, show a specific band.Wherein: M is molecular weight Marker; 1 is the amplified band take the Klebsiella pneumonia genomic dna as template; 2 negative contrasts.
4, reclaim pcr amplification product and checking order, sequencing result shows, the size of pcr amplification product is 558bp.
Respectively each citrus fruit fly sample (sample 1 is to sample 17) is carried out following evaluation:
1, extracts genomic dna
The blood of employing sky, Beijing root biochemical technology company limited/cell/tissue genome DNA extracting reagent kit extracts the genomic dna of the whole polypide of single head citrus fruit fly adult.
2, PCR identifies
The genomic dna that extracts take step 1 is as template (using water as negative contrast), and the special primer that designs with the step 2 of embodiment 1 obtains pcr amplification product to carrying out pcr amplification.
Pcr amplification volume (50 μ L): 5 μ L, 10 * Reaction Buffer (contains Mg
2+), 4 μ L dNTP mixture (2.5mM), Taq enzyme 0.5 μ L (2.5U/ μ L, sky, Beijing root biochemical technology company limited), ddH
2O 37.5 μ L, genomic dna 1 μ L (115.8ng/ μ L), upstream primer (10 μ M) 1 μ L, downstream primer (10 μ M) 1 μ L.
Pcr amplification condition: 94 ℃ of 3min; 94 ℃ of 30sec, 59.6 ℃ of 30sec, 72 ℃ of 30sec, 35 circulations; 72 ℃ of 5min.
Get 5 μ L pcr amplification products, carry out 1.5% agarose gel electrophoresis and ethidium bromide (EB) dyeing, in gel imaging system, observe and imaging (Gel Logical Pro 212).
The results are shown in Figure 2 (band between the 500bp-700bp is the purpose band), M is DNA relative molecular weight standard, and swimming lane 1 to swimming lane 17 is followed successively by sample 1 to 17, swimming lane 18 negative contrasts.Sample 1 to 9, sample 10, sample 12 and sample 16 all show to have the purpose band, and preliminary judgement contains Klebsiella pneumonia.
3, order-checking is identified
Reclaim respectively each purpose band and check order, the size of the purpose band of sample 1 to 9 and sample 16 is 558bp.The purpose band of sample 10 and sample 12 a little less than, reclaim concentration lower, do not obtain sequencing result.Sample 1 is to sample 8 consistent with the sequencing result of sample 16 (seeing the sequence 3 of sequence table), the online comparison result shows of NCBI, with GENBANK ACCESSION NO. be that the sequence similarity of HE578781.1 is the highest, be 100%.The sequencing result of sample 9 is seen the sequence 4 of sequence table, the online comparison result shows of NCBI, with GENBANK ACCESSION NO. be that the sequence similarity of JN625720.1 is the highest, be 100%.
4, Morphology and physiology biochemical identification
The Morphology and physiology biochemical character of Klebsiella pneumonia is as follows: be shaped as long direct rod shape, diameter is 0.3-1.0 μ m, but Individual existence or arrange with paired, short chain shape, has the pod membrane that Polysaccharides (polysaccharide) forms, have the pili little than flagellum (fimbriae) around the thalline, can help it to stick, not have mobility; Gram-negative bacteria, amphimicrobian; Be opaque light yellow bacterium colony at the PCA substratum.
Respectively sample 1 to sample 17 is carried out following experiment: with the abrasive material of citrus fruit fly coating PCA culture medium flat plate, with single bacterium colony switching spread plate 3 times, obtain to carry out the Morphology and physiology biochemical character after the pure culture of bacterium and identify.Sample 1 to 9 has all obtained to have the Klebsiella pneumonia of above-mentioned morphological specificity and physiological and biochemical property in sample 10, sample 12 and the sample 16, do not have to obtain to have the Klebsiella pneumonia of above-mentioned morphological specificity and physiological and biochemical property in other sample.
Experiment sample: 10 of the female worms of citrus fruit fly that Inspection and Quarantine Technic Center, Guangdong Entry-Exit Inspection and Qu raises, collection is from 10 of the citrus fruit fly male worms of Thailand, collection is from 10 of the citrus fruit fly male worms of Shanghai Baoshan, collection is from 10 of the citrus fruit fly male worms of Qingpu Shanghai, collection is from 10 of the citrus fruit fly male worms of Bay, Ningde, Fujian Province, collection is from 10 of the citrus fruit fly male worms of Foochow, Fujian, collection is from 10 of the citrus fruit fly male worms of Qionghai, collection is from 10 of the citrus fruit fly male worms of Shanghai International Automobile City Tourist Festival, collection is from 10 of the citrus fruit fly male worms in Songjiang, Shanghai, collection is from 10 of the citrus fruit fly male worms of Ningbo of Zhejiang, collection is from 10 of the citrus fruit fly male worms of Xiamen of Fujian Province, collection is from 10 of the citrus fruit fly male worms of Guangzhou Guangdong, collection is from 10 of the citrus fruit fly male worms of Pingxiang, Guangxi, collection is from 10 of the citrus fruit fly male worms of Jinghong In Yunnan Province, collection is from 10 of the citrus fruit fly male worms of Xingyi of Guizhou, collection is from 10 of the citrus fruit fly male worms of Nanning, collection is from 10 of the female worms of the citrus fruit fly of Qionghai, totally 180 citrus fruit fly samples.
Respectively each experiment sample is pressed the method for the step 1 of embodiment 3 and extracted genomic dna, and carry out PCR by the method for the step 2 of embodiment 3 and identify, show that the sample of the band between the 500bp-700bp is for infecting the sample of citrus fruit fly.In 180 citrus fruit flies, have 133 citrus fruit flies to contain Klebsiella pneumonia, infection rate is 73.9%.
The sensitivity test experience of embodiment 5, primer pair
1, adopt bacterial genomes DNA extraction test kit (sky, Beijing root biochemical technology company limited) to extract the genomic dna of Klebsiella pneumonia.
2, detect the concentration of genomic dna, it is carried out serial dilution with the TE damping fluid, obtain 8 kinds of diluents; Genomic dna concentration in 8 kinds of diluents is respectively: 40ng/ μ L, 20ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L, 0.0001ng/ μ L, 0.00001ng/ μ L and 0.000001ng/ μ L are followed successively by diluent 1 to diluent 10.
3, (use water as negative contrast) take every kind of diluent as template respectively, to carrying out pcr amplification, obtain pcr amplification product with the special primer of the step 2 of embodiment 1 design.
Pcr amplification volume (50 μ L): 5 μ L, 10 * Reaction Buffer (contains Mg
2+), 4 μ L dNTP mixture (2.5mM), Taq enzyme 0.5 μ L (2.5U/ μ L, sky, Beijing root biochemical technology company limited), ddH
2O 37.5 μ L, diluent (containing genomic dna) 1 μ L, upstream primer (10 μ M) 1 μ L, downstream primer (10 μ M) 1 μ L.
Pcr amplification condition: 94 ℃ of 3min; 94 ℃ of 30sec, 59.6 ℃ of 30sec, 72 ℃ of 30sec, 35 circulations; 72 ℃ of 5min.
4, get 5 μ L pcr amplification products, carry out 1.5% agarose gel electrophoresis and ethidium bromide (EB) dyeing, in gel imaging system, observe and imaging (Gel Logical Pro 212).
The electrophoresis result of the pcr amplification product of each diluent is seen Fig. 3.Among Fig. 3, M:DNA relative molecular weight standard (D2000); Swimming lane 1 to swimming lane 10 is followed successively by diluent 1 to diluent 10,11 negative contrasts.The result shows, the right sensitivity of this special primer is higher, and can detect minimum template amount is 0.01ng.After primer concentration arrived certain altitude, band brightness did not increase with the rising of concentration.
Claims (7)
1. the special primer of assistant identification Klebsiella pneumonia pair is comprised of DNA shown in the sequence 2 of DNA shown in the sequence 1 of sequence table and sequence table.
2. the described special primer of claim 1 is to the application in the test kit of preparation assistant identification Klebsiella pneumonia.
3. the test kit of an assistant identification Klebsiella pneumonia comprises the described special primer of claim 1 pair.
4. whether the described special primer of claim 1 is to carrying the application in the Klebsiella pneumonia at the assistant identification citrus fruit fly.
5. the described special primer of claim 1 is to the application in the test kit that whether carries Klebsiella pneumonia at preparation assistant identification citrus fruit fly.
6. whether an assistant identification citrus fruit fly carries the test kit of Klebsiella pneumonia, comprises the described special primer of claim 1 pair.
7. method whether the assistant identification citrus fruit fly carries Klebsiella pneumonia, comprise the steps: that genomic dna take citrus fruit fly to be measured is as template, with the described special primer of claim 1 to carrying out pcr amplification, carry Klebsiella pneumonia if obtain the described citrus fruit fly of pcr amplification product, do not carry Klebsiella pneumonia if obtain the described citrus fruit fly of pcr amplification product.
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| CN104531887B (en) * | 2015-01-15 | 2016-08-17 | 王贵升 | The authentication method of Klebsiella pneumonia and the primer |
| CN104946762B (en) * | 2015-06-17 | 2018-02-06 | 中生北控生物科技股份有限公司 | Detect the kit of Friedlander's bacillus |
| CN105132555B (en) * | 2015-09-08 | 2018-05-29 | 重庆文理学院 | The PCR detection kit and detection method of a kind of pneumonia infection Klebsiella of Chinese giant salamander, Andrias davidianus |
| CN111073988B (en) * | 2020-01-08 | 2022-05-31 | 华南农业大学 | PCR detection primers for pathogen Klebsiella of mulberry bacterial wilt and application |
| CN112226526A (en) * | 2020-10-12 | 2021-01-15 | 河南省奶牛生产性能测定有限公司 | Milk cow mastitis Klebsiella pneumoniae detection kit and detection method |
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| CN101892300B (en) * | 2010-02-10 | 2012-09-05 | 广州华峰生物科技有限公司 | Klebsiella pneumoniae detection kit and use method thereof |
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| CN109486778B (en) * | 2018-10-22 | 2020-08-25 | 浙江科技学院 | Co-evolution network-based omega-transaminase mutant and preparation method and application thereof |
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