CN103343168B - Rapid identification method for pathogenic fungi - Google Patents
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Abstract
The invention relates to a rapid identification method for pathogenic fungi, which specially comprises the following steps: (1) culturing fungi on a solid culture medium; (2) taking fresh fungi, and extracting the DNA of the fungi by using a microwave method; (3) amplifying DNA fragments between ribosomal spacers of the fungi by using ITS1 and ITS4 primers through PCR (polymerase chain reaction); (4) after recovering the PCR fragments, carrying out sequence determination on the fragments; and (5) carrying out comparison on the obtained sequences by using the Blast of NCBI. According to the invention, the identification of unknown pathogenic fungi can be performed within a shorter time and under relatively simple experimental conditions, thereby facilitating the rapid diagnosis for diseases occurring in fields so as to guide the prevention and control of diseases. According to the method, through the identification on 21 kinds of pathogenic fungi on different crops, and the coincidence rate is 100% through sequence comparison. The method is not only applicable to the identification on fresh mycelia, but also is applicable to the identification on conidium and sclerotium of pathogenic fungi, and has the characteristics of rapidness and high efficiency.
Description
Technical field
The invention belongs to Identification of The Fungal Species Causing field, be specifically related to a kind of method of Rapid identification pathogenic fungi.
Background technology
Peanut pathogenic fungi harm peanut causes production loss, affects the quality of product.In order to effective controlling disease, need the cause of disease of Rapid identification peanut, timely specific aim prevents and treats this disease.At present to fungi except traditional Morphological Identification, in order to obtain information more accurately, general dependence, to the understanding of fungal genomic DNA information, carries out sequential analysis by the PCR primer obtained after the PCR primer pair fungal gene group nrDNA ITS amplification that fungi is general.The extracting method of common fungus DNA is: liquid culture 5-7d is obtained hypha,hyphae group and filter, the broken hypha,hyphae cell walls of CTAB or SDS method is passed through after drying or liquid nitrogen freezing process, remove albumen by benzene/chloroform again, eventually pass Virahol or alcohol settling obtains fungal DNA.The fungal DNA obtained is obtained goal gene fragment through pcr amplification, be connected in carrier T by reclaiming goal gene fragment after gel electrophoresis again, and transform bacteria competent cell, the mono-clonal that the competent cell spread plate transformed obtains after cultivating is accredited as positive colony by PCR, and the bacterium liquid of positive colony after cultivating is submitted to and carried out sequential analysis.In this traditional method, the time of fungus culture is long, need the amount of hypha,hyphae more, the method spended time that fungal DNA extracts is longer, and the reagent expended and human cost are also higher, complex steps, fungal DNA by connection carrier after pcr amplification, transform bacteria competence, the clone of acquisition carries out identifying also needs at least 2 days time, add the follow-up order-checking time, traditional authentication method at least needs the time of about 2 weeks.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of simple to operate, cost is low, flux is high and be applicable to the authentication method of Rapid identification pathogenic fungi.
For realizing goal of the invention, technical scheme of the present invention is:
A method for Rapid identification pathogenic fungi, comprises the steps:
(1) cultivate fungi to be identified, obtain radicula byssoidea, sclerotium or conidium;
(2) get above-mentioned radicula byssoidea, sclerotium or conidium and be placed in damping fluid, carry out microwave thermal and shake after broken wall treatment, be placed on ice, after centrifugal, get supernatant liquor, be fungal DNA extracting solution;
(3) utilize fungi universal primer ITS1 and ITS4 to carry out pcr amplification to above-mentioned fungal DNA extracting solution, amplified production carries out agarose gel electrophoresis, and recycling PCR reclaims test kit recovery purifying and obtains object fragment;
(4) above-mentioned purpose fragment is directly carried out sequential analysis, the nucleotides sequence obtained is listed in NCBI website use blast and searches for the fungal sequence matched with it, and matching length is the longest, and the fungi that sequence identity is 100% is judged to be the fungi belonging to identified fungi.
In such scheme, described fungi to be identified is selected from step (1): aspergillus flavus bacterium, Sclerotium rolfssi, peanut focal spot germ, peanut reaping hook germ, peanut alternaric bacteria, apple sheath blight fungus, Fulvia fulva, gaeumannomyces graminis, lawn coin pinta bacterium, verticillium dahliae, Didymella bryoniae, Ustilaginoidea virens, dosporium cucumerinumand its, maize Curvularia leaf spot fungi, sweet potato black rot pathogen, rice blast fungus, cucumber fusarium axysporum, hami melon wilt, Alternaria brassicae, Chinese sorghum rhizoctonia solani, citrus scab bacterium, Chinese ephedra beading reaping hook fungi, Colletotrichum capsici, cucumber anthracnose, or cereal reaping hook fungi.
In such scheme, step (1) described fungi carries out flat board or slant culture on PDA substratum or czapek's solution.
In such scheme, step (1) described fungi 28 DEG C of light culture on PDA substratum grow to 2 ~ 3cm to bacterium colony in 1 ~ 5 day, obtain described radicula byssoidea; Or described fungi 28 DEG C of dark culturing 7 ~ 15 days on PDA substratum, obtain described sclerotium; Or described fungi 28 DEG C of dark culturing 5 ~ 7 days on czapek's solution, obtain described conidium.
In such scheme, step (2) described radicula byssoidea, sclerotium or conidial quality are 0.2 ~ 2mg.
In such scheme, step (2) described damping fluid is TE damping fluid, and its moiety is: 10mMTris-HCl, 1mM EDTA, pH=8.0.
In such scheme, the shake microwave power of broken wall treatment of step (2) described microwave thermal is 750W, and the time of microwave treatment is 30s ~ 5min.
In such scheme, step (2) described microwave thermal is shaken after broken wall treatment, is placed in 5 ~ 10min on ice immediately, then carries out centrifugal.
In such scheme, describedly centrifugally at room temperature to carry out, centrifugal rotating speed is 10000rpm, and the centrifugal time is 1 ~ 2min.
The system of above-mentioned pcr amplification is: the ITS1 primer of 10 × Taq buffer2 μ l, 10pmol and each 1 μ l of ITS4 primer, fungal DNA extracting solution 2 μ l, 1U Taq DNA polymerase1 μ l, 2.5mM MgCl
21 μ l and 2mmol/L dNTPs1 μ l, then complement to 20 μ l with aqua sterilisa; Amplification condition is: 94 DEG C of 3min sex change, 94 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min, 35 circulations, and 72 DEG C of 7min extend.
The ultimate principle that the present invention utilizes is: utilize microwave thermal to shake the hypha,hyphae of broken wall treatment trace, fungal DNA can discharge and enter damping fluid from cell; Can passivation DNA enzymatic under EDTA in damping fluid TE and low temperature environment, centrifuging removes fungal cell's relic and enzyme; The supernatant liquor obtained is as the template of PCR, and the PCR primer ITS1 utilizing fungi general and ITS4 increases to district between fungi rrna, and the PCR primer obtained directly carries out sequencing after reclaiming; By carrying out the highest fungal DNA sequence of Blast comparison consistence to the sequence measured in NCBI sequence library, be the fungi belonged to targeted fungal.In the present invention, the whole qualification completing fungi only needs the time of 1 week.
Beneficial effect of the present invention: the present invention is simple to operate, cost is low, flux is high, qualification speed is fast, reliability is high; Compared with traditional method, present method has saved mirror time, reagent, consumptive material and manpower, is applicable to carrying out quick diagnosis to peanut and other crop cause of diseases, and the time has been striven in the control for fungal disease.
Accompanying drawing explanation
Fig. 1 is the pcr amplification result of the DNA that peanut focal spot mycelia obtains under the different TE concentration different microwave action time, the band 1:30S10 × TE in figure, 2:30S5 × TE, 3:30S1 × TE, 4:30s ddH
2o, 5:1min10 × TE, 6:1min5 × TE, 7:1min1 × TE, 8:1min ddH2O, 9:2min10 × TE, 10:2min5 × TE, 11:2min1 × TE, 12:2min ddH2O, 13:3min10 × TE, 14:3min5 × TE, 15:3min1 × TE, 16:3min ddH2O, 17:5min10 × TE, 18:5min5 × TE, 19:5min1 × TE, 20:5min ddH2O, 21CK(clear water contrasts).
Fig. 2 is the pcr amplification result of the pathogenic fungi DNA of the Different Crop adopting multistage microwave amplifier, band 1 in figure: apple sheath blight fungus, 2: Fulvia fulva, 3: gaeumannomyces graminis, 4: lawn coin pinta bacterium, 5: verticillium dahliae, 6: Didymella bryoniae, 7: paddy rice rice district germ, 8: dosporium cucumerinumand its, 9: maize Curvularia leaf spot fungi, 10: sweet potato black rot pathogen, 11: rice blast fungus, 12: cucumber fusarium axysporum, 13: peanut focal spot germ, 14: hami melon wilt, 15: Alternaria brassicae, 16: Chinese sorghum rhizoctonia solani, 17: citrus scab bacterium, 18: Chinese ephedra beading reaping hook fungi, 19: Colletotrichum capsici, 20: cucumber anthracnose, 21: cereal reaping hook fungi, 22:CK(clear water contrasts).
Fig. 3 is the pcr amplification result of the different shape peanut pathogenic fungi DNA adopting multistage microwave amplifier, band 1 in figure, 2 is from different local peanut focal spot bacterium, 3: aspergillus flavus spore, 4: peanut alternaric bacteria, 5,6,7 is from different local peanut sickle-like bacteria, 8: the white thin,tough silk mycelia of peanut, 9: the white thin,tough silk sclerotium of peanut.
Embodiment
The present invention is in order to obtain the processing parameter of multistage microwave amplifier fungal DNA, and with peanut focal spot bacterium mycelia for research object, have studied the Parameter Conditions of multistage microwave amplifier fungal DNA, concrete operation step is as follows:
(1) peanut focal spot bacterium is cultivated: be inoculated on PDA substratum by peanut focal spot bacterium, 28 DEG C of light culture 4d grow to 2 ~ 3cm to bacterium colony, obtain hypha,hyphae.
(2) multistage microwave amplifier fungal DNA: utilize inoculating needle to be got in the centrifuge tube of 0.2mg to the 1.5ml of sterilizing by the fresh mycelia (i.e. hypha,hyphae) that PDA substratum grows, adds 50 μ l10 × TE damping fluids, 50 μ l5 × TE damping fluids, 50 μ l1 × TE damping fluids or 50 μ l H
2o, after shaking several seconds, is positioned in microwave oven, 750W microwave 30s, 1min, 2min, 3min, or 5min, be placed in 5min on ice immediately after taking-up, then get supernatant liquor after centrifugal (centrifugal rotational speed is 10000rpm, and the time is 1min) and transfer in fresh centrifuge tube, obtain fungal DNA extracting solution.The component of described TE damping fluid is: 10mM ris-HCl, 1mM EDTA, pH=8.0.
(3) utilize fungi universal primer ITS1 and ITS4 to carry out pcr amplification to above-mentioned fungal DNA extracting solution, the system of pcr amplification is: the ITS1 primer of 10 × Taq buffer2 μ l, 10pmol and each 1 μ l of ITS4 primer, fungal DNA extracting solution 2 μ l, 1U Taq DNA polymerase1 μ l, 2.5mM MgCl
21 μ l, 2mmol/L dNTPs1 μ l, aqua sterilisa complement to 20 μ l; Amplification condition is: 94 DEG C of 3min sex change, 94 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min, 35 circulations, and 72 DEG C of 7min extend.
(4) amplified production carries out agarose gel electrophoresis, the results are shown in following table 1 and Fig. 1.
Can obtain from table 1 and Fig. 1: the fresh mycelia 0.2mg of fungi of trace is placed in TE damping fluid (1 × TE damping fluid ~ 10 × TE damping fluid), carry out short period of time microwave treatment (30s ~ 5min) all can obtain enough DNA and carry out pcr amplification, and amplified band is clear, illustrate that the method is applicable to the Rapid identification of pathogenic fungi.
Low-concentration buffer is little on follow-up PCR reaction impact, and the supernatant liquor after microwave directly as the template of PCR reaction, can decrease treatment step and the loss of DNA.The condition that the present invention chooses " concentration 1 × TE damping fluid; microwave action time 30s ~ 5min " is as the condition of multistage microwave amplifier fungal DNA in following embodiment 1 and embodiment 2, and employing multistage microwave amplifier fungal DNA can save the time of extraction required for fungal DNA, manpower and reagent consumption greatly.
The pcr amplification result of the DNA sample that the different TE buffer concentration of table 1, different treatment time obtain
| Treatment time | 10XTE | 5XTE | 1XTE | H 2O |
| 30s | + | + | + | + |
| 1min | + | + | + | - |
| 2min | + | + | + | - |
| 3min | + | + | + | - |
| 5min | + | + | + | - |
Note: "+" expression obtains target DNA fragment; "-" expression does not amplify target DNA fragment
Embodiment 1
A method for Rapid identification pathogenic fungi, specifically comprises the steps:
(1) cultivate fungi to be identified: be inoculated into by fungi to be identified on PDA substratum, 28 DEG C of light culture grow to 2-3cm to bacterium colony, obtain radicula byssoidea.
(2) DNA of multistage microwave amplifier fungi: utilize inoculating needle to get in the centrifuge tube of 2mg to the 1.5ml of sterilizing by the fresh mycelia that PDA substratum grows (i.e. above-mentioned radicula byssoidea), 1 × TE damping fluid 50 μ the l added, shake after several seconds, be positioned in microwave oven, 750W microwave 2min, is placed in 7min on ice after taking-up immediately, more centrifugal (centrifugal rotational speed is 10000rpm, time is 1.5min) after get supernatant liquor and transfer in fresh centrifuge tube, namely obtain fungal DNA extracting solution.
(3) utilize fungi universal primer ITS1 and ITS4 to carry out pcr amplification to fungal DNA extracting solution, the system of pcr amplification is: the ITS1 primer of 10 × Taq buffer2 μ l, 10pmol and each 1 μ l of ITS4 primer, fungal DNA extracting solution 2 μ l, 1U Taq DNA polymerase1 μ l, 2.5mM MgCl
21 μ l, 2mmol/LdNTPs1 μ l, aqua sterilisa complement to 20 μ l; Amplification condition is: 94 DEG C of 3min sex change, 94 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min, 35 circulations, and 72 DEG C of 7min extend.
(4) PCR primer direct sequence measures: amplified production is carried out agarose gel electrophoresis, and recycling sky root PCR reclaims test kit and reclaims object fragment, delivers Shanghai Invitrogen company and carries out sequential analysis.
(5) NCBI blast sequence, judge which kind of fungi unknown fungi is: the nucleotides sequence obtained is listed in NCBI website use blast and searches for the fungal sequence matched, matching length is the longest, and the fungi of sequence identity 100% is judged to be the fungi belonging to unknown fungi.
Above-mentioned fungi to be identified is selected from: apple sheath blight fungus, Fulvia fulva, gaeumannomyces graminis, lawn coin pinta bacterium, verticillium dahliae, Didymella bryoniae, Ustilaginoidea virens, dosporium cucumerinumand its, maize Curvularia leaf spot fungi, sweet potato black rot pathogen, rice blast fungus, cucumber fusarium axysporum, peanut focal spot germ, hami melon wilt, Alternaria brassicae, Chinese sorghum rhizoctonia solani, citrus scab bacterium, Chinese ephedra beading reaping hook fungi, Colletotrichum capsici, cucumber anthracnose, or cereal reaping hook fungi,
The incubation time of above-mentioned 21 kinds of fungies be 1 ~ 5 day not etc.
2, the Fig. 2 that the results are shown in Figure of pcr amplification describes the Rapid identification that present method is applicable to above-mentioned Different Crop pathogenic fungi.
The sequence alignment of above-mentioned 21 kinds of fungies the results are shown in following table 2, and result shows, the reliability of the method for the Rapid identification pathogenic fungi of the present embodiment reaches 100%.
The result of table 2 Different Crop pathogenic bacteria Rapid identification
| Crop pest | Pathogenic bacteria | PCR | Sequence blast |
| Apple sheath blight fungus | Physalospora piricola | + | + |
| Fulvia fulva | Fulria fulva | + | + |
| Gaeumannomyces graminis | Gaeumannomyces graminis | + | + |
| Lawn coin pinta bacterium | Moellerodiscus spp | + | + |
| Verticillium dahliae | Verticillium dahliae | + | + |
| Didymella bryoniae | Didymella Bryoniae | + | + |
| Ustilaginoidea virens | Ustilaginoidea virens | + | + |
| Dosporium cucumerinumand its | Cladosporium cucumerinum | + | + |
| Maize Curvularia leaf spot fungi | Curvularia lunata | + | + |
| Sweet potato black rot pathogen | Ceratocystis fimbriata | + | + |
| Rice blast fungus | Magnaporthe oryzae | + | + |
| Cucumber fusarium axysporum | Fusarium oxysporum.sp.cucumebrium | + | + |
| Peanut focal spot germ | Leptosphaerulina crassiasca | + | + |
| Hami melon wilt | Fusarium oxysporum f.sp.Melonis | + | + |
| Alternaria brassicae | Alternaria brassicae | + | + |
| Chinese sorghum rhizoctonia solani | Rhizoctonia solani Kühn | + | + |
| Citrus scab bacterium | Sphaceloma fawcetti Jenk | + | + |
| Chinese ephedra beading reaping hook fungi | Fusarium moniliforme | + | + |
| Colletotrichum capsici | Colletotrichum capsici | + | + |
| Cucumber anthracnose | Colletotrichum lagenarium | + | + |
| Cereal reaping hook fungi | Fusarium graminearum Schw | + | + |
| Clear water contrasts | - |
Note: "+" expression obtains object PCR fragment; "-" expression does not amplify object fragment
Embodiment 2
The qualification object of the present embodiment comprises hypha,hyphae (radicula byssoidea), sclerotium and conidium, comprises three key steps such as the extraction of fungal DNA, the amplification of PCR specific fragment and the sequencing of amplified fragments.
A kind of method of Rapid identification pathogenic fungi, concrete operation step is roughly the same with embodiment 1, difference is: (1) cultivates fungi to be identified: be inoculated on PDA substratum respectively by the Aspergillus flavus that peanut is separated, focal spot bacterium, alternaric bacteria, sickle-like bacteria and white thin,tough silk bacterium, 28 DEG C of light culture 1 ~ 4 day are not etc., grow to 2-3cm to bacterium colony, obtain the mycelia (i.e. radicula byssoidea) of Aspergillus flavus, focal spot bacterium, alternaric bacteria, sickle-like bacteria and white thin,tough silk bacterium; White for peanut thin,tough silk bacterium to be inoculated on PDA substratum 28 DEG C of light culture obtain the white thin,tough silk bacterium of peanut sclerotium to 10d simultaneously; Aspergillus flavus bacterium to be inoculated on czapek's solution (CZA) substratum the conidium that 28 DEG C of dark culturing 5d obtain aspergillus flavus.
(2) multistage microwave amplifier fungal DNA: get fresh mycelia, sclerotium or conidium 1mg in the centrifuge tube of the 1.5ml of sterilizing, 1 × TE damping fluid 50 μ the l added, shake after several seconds, be positioned in microwave oven, 750W microwave 2min, places 10min on ice after taking-up fast, more centrifugal (centrifugal rotational speed is 10000rpm, time is 2min) after get supernatant liquor and transfer in fresh centrifuge tube, namely obtain fungal DNA extracting solution.
The form of above-mentioned fungi sees the following form 3, and 3, the Fig. 3 that the results are shown in Figure of pcr amplification describes the DNA rapid extraction that the method is applicable to the mycelia of peanut various pathogenic bacteria, conidium or sclerotium.
Following table 3 be the results are shown in the sequence alignment of above-mentioned 5 kinds of fungies, result shows, the micro-fresh mycelia of fungi, sclerotium or conidium, at lower concentration 1 × TE damping fluid, after short period of time microwave treatment, all can obtain enough DNA and carry out pcr amplification, pcr amplification band is clear, sequence blast comparison shows, and the reliability of the method for the Rapid identification pathogenic fungi of the present embodiment reaches 100%.
The qualification result of table 3 Different Kinds of Pathogens hypha,hyphae, conidium and sclerotium
| Pathogenic bacteria | Form | PCR | Sequencing |
| Aspergillus flavus bacterium | Conidium | + | + |
| Peanut focal spot bacterium | Mycelia | + | + |
| Peanut alternaric bacteria | Mycelia | + | + |
| Peanut sickle-like bacteria | Mycelia | + | + |
| Sclerotium rolfssi | Mycelia | + | + |
| Sclerotium rolfssi | Sclerotium | + | + |
Note :+obtain object PCR fragment;-expression does not amplify object fragment
Obviously, above-described embodiment is only for the example done clearly is described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And therefore amplified apparent change or variation are still within the protection domain of the invention.
Claims (8)
1. a method for Rapid identification pathogenic fungi, is characterized in that comprising the steps:
(1) cultivate fungi to be identified, obtain radicula byssoidea, sclerotium or conidium;
(2) get above-mentioned radicula byssoidea, sclerotium or conidium and be placed in damping fluid, carry out microwave thermal and shake after broken wall treatment, be placed on ice, after centrifugal, get supernatant liquor, be fungal DNA extracting solution;
(3) utilize fungi universal primer ITS1 and ITS4 to carry out pcr amplification to above-mentioned fungal DNA extracting solution, amplified production carries out agarose gel electrophoresis, and recycling PCR reclaims test kit recovery purifying and obtains object fragment;
(4) above-mentioned purpose fragment is directly carried out sequential analysis, the nucleotides sequence obtained is listed in NCBI website use blast and searches for the fungal sequence matched with it, matching length is the longest, and the fungi that sequence identity is 100% is judged to be the fungi belonging to identified fungi;
Described damping fluid is TE damping fluid, and its moiety is: 10mM Tris-HCl, 1mM EDTA, pH=8.0; The shake microwave power of broken wall treatment of described microwave thermal is 750W, and the time of microwave treatment is 30s ~ 5min.
2. method according to claim 1, it is characterized in that the described fungi to be identified of step (1) is selected from: aspergillus flavus bacterium, Sclerotium rolfssi, peanut focal spot germ, peanut reaping hook germ, peanut alternaric bacteria, apple sheath blight fungus, Fulvia fulva, gaeumannomyces graminis, lawn coin pinta bacterium, verticillium dahliae, Didymella bryoniae, Ustilaginoidea virens, dosporium cucumerinumand its, maize Curvularia leaf spot fungi, sweet potato black rot pathogen, rice blast fungus, cucumber fusarium axysporum, hami melon wilt, Alternaria brassicae, Chinese sorghum rhizoctonia solani, citrus scab bacterium, Chinese ephedra beading reaping hook fungi, Colletotrichum capsici, cucumber anthracnose, or cereal reaping hook fungi.
3. method according to claim 1, is characterized in that step (1) described fungi carries out flat board or slant culture on PDA substratum or czapek's solution.
4. method according to claim 1, is characterized in that step (1) described fungi 28 DEG C of light culture on PDA substratum grow to 2 ~ 3cm to bacterium colony in 1 ~ 5 day, obtains described radicula byssoidea; Or described fungi 28 DEG C of dark culturing 7 ~ 15 days on PDA substratum, obtain described sclerotium; Or described fungi 28 DEG C of dark culturing 5 ~ 7 days on czapek's solution, obtain described conidium.
5. method according to claim 1, is characterized in that step (2) described radicula byssoidea, sclerotium or conidial quality are 0.2 ~ 2mg.
6. method according to claim 1, is characterized in that step (2) described microwave thermal is shaken after broken wall treatment, is placed in 5 ~ 10min on ice immediately, then carries out centrifugal.
7. method according to claim 1, it is characterized in that described centrifugally at room temperature to carry out, centrifugal rotating speed is 10000rpm, and the centrifugal time is 1 ~ 2min.
8. method according to claim 1, is characterized in that the system of described pcr amplification is: the ITS1 primer of 10 × Taq damping fluid 2 μ l, 10pmol and each 1 μ l of ITS4 primer, fungal DNA extracting solution 2 μ l, 1UTaq archaeal dna polymerase 1 μ l, 2.5mM MgCl
21 μ l, 2mmol/L dNTPs 1 μ l and aqua sterilisa complement to 20 μ l; Amplification condition is: 94 DEG C of 3min sex change, 94 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min, 35 circulations, and 72 DEG C of 7min extend.
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