CN104862404A - Multiple PCR (Polymerase Chain Reaction) detection kit, special primer and multiple PCR detection method for two kinds of seed-borne diseases on rice - Google Patents
Multiple PCR (Polymerase Chain Reaction) detection kit, special primer and multiple PCR detection method for two kinds of seed-borne diseases on rice Download PDFInfo
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Abstract
The invention discloses a multiple PCR (Polymerase Chain Reaction) detection kit, a special primer and a multiple PCR detection method for two kinds of seed-borne diseases on rice. The two kinds of seed-borne diseases are xanthomonas oryzae pv.oryzae and xanthomonas oryzae pv.oryzicola. A pair of special primers X-b for detecting the xanthomonas oryzae pv.oryzae and a pair of special primers X-t for detecting the xanthomonas oryzae pv.oryzicola are designed for the kit and the method, wherein the fragment length of a target product of each primer X-b is 461 bp; the fragment length a target product of each primer X-t is 348 bp; when the causes of the two kinds of diseases are detected at the same time, a multiple PCR reaction system as well as reaction conditions are optimized. According to the multiple PCR detection kit, the special primer and the multiple PCR detection method disclosed by the invention, the detection procedures of the causes of the two kinds of diseases are detected at the same time, the operation is simple and convenient, and quick and accurate, the cost is lower, and cloning, sequencing and sequence alignment are not required.
Description
Technical field
The present invention relates to the technical field of molecular biological detection of two kind of plant cause of diseases, be specifically related to the multiple molecular biological detection method of rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) and Xanthomonas campestris PV.oryzicola (Xanthomonas oryzaepv.oryzicola).
Background technology
China is the fourth-largest importer of Agricultural Products Trade and the fifth-largest export State, the further expansion of China's Foreign Trade is made under the trend of economic globalization, the allocation and transportation of plants and plant product are increased, the invasion simultaneously also bringing comparatively serious harmful organism threatens, the problem that this oneself through becoming harm China species diversity, ecotope and national economy one is very important and urgent.Nowadays, the rice paddy seed industrial market of China is compared compared to the main Rice Production state such as South East Asia and South America, and advantage is comparatively obvious, no matter variety classification or the methods of conducting trade are tending towards variation all gradually.But due to the driving of economic interests, some illegal retailers produce privately, manage rice paddy seed.This not only serious quality and prestige compromising China's seed export, result also in of poor quality, and band poison or the seed being not suitable for ecological region planting condition come into the market.Each entry and exit port and place of production phytopathological inspection quarantine an urgent demand efficient and sensible, fast and simple diagnostic techniques and instrument, to strengthen port quarantine, construct the first barrier taking precautions against the invasion of external epidemic disease evil, to guarantee China agricultural production security, outlet and the internal trade of serving agricultural-food are of great immediate significance.
On paddy rice of the present invention, two kinds of seed-borne diseases refer to the bacterial blight of rice caused by rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) respectively and the bacterial leaf streak of rice caused by xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola).
Bacterial blight of rice is the important quarantine seed-borne disease of a kind of paddy rice caused by rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae).Its thalline is rod-short, unit cell, single flagellum, and extremely raw or Asia extremely life, embraces without bud, and without capsule, the exocellular polysaccharide of the outer tool cement of thalline surrounds, and Gram-negative is aerobic.Bacterium colony honey gold or faint yellow on gravy peptone nutrient agar substratum, circular, periphery is neat, and homogeneous, surface elevation, light is along shinny, and viscosity, produces non-water-soluble yellow pigment.Pathogen growth optimum temperuture is 25-30 DEG C, minimum 5 DEG C, the highest 40 DEG C, adhesive-free film protection under 53 DEG C 10 minutes lethal, have glued membrane protect lower 57 DEG C 10 minutes lethal.Germ optimum potential of hydrogen is pH6.5-7.0.
This kind of germ also known as white leaf pest, burn, cogongrass pest etc.The whole breeding time of paddy rice all can be killed, and especially seedling stage, a point fringe phase are injured the heaviest, and each organ all can be caught an illness, and blade is the most easily caught an illness.And this germ not only can be attached to seed-coat sometimes also can be deposited with Interior Seed.Its symptom has larger difference, the common withered type of leaf that mainly contains, acute wilting type, foxiness or brown stain type 3 type because of germ infestation position, variety resistance, envrionment conditions:
(1) the withered type of leaf, mainly to cause harm blade, also to cause harm time serious leaf sheath, first, from blade tip or leaf margin, first there is sap green water soaking mode wire spot in morbidity, very fast along wire spot formation yellow-white scab, then along leaf margin both sides or middle rib expansion, become tawny, finally in withered white, scab edge boundary is obvious.
(2) acute wilting type, from seedling stage to a point evil phase, susceptible disease when germ invades vascular bundle from root system or basal part of stem wound.And stem is fallen ill simultaneously, lobus cardiacus dehydration green grass or young crops is withered, wilting death, and rest blade is also successively blue or green withered curling, and then complete stool is withered, also has the situation that only lobus cardiacus is withered.
(3) foxiness or brown stain type, more common on disease-resistant variety, and germ is invaded by artificial leaf-cutting or wound, and the low or unfavorable onset condition at temperature, when brown necrotic reaction band appears in scab periphery, can stagnate expansion.
Yellow type symptom is rarer, and its main manifestations in early days lobus cardiacus is not withered, and can produce irregular chlorisis spot, and catching an illness, the later stage can develop into withered and yellow spot.
This germ is as the important quarantine seed-borne disease of a kind of paddy rice, and harm is only second to bacterial leaf streak of rice, and in China except Xinjiang, Tibet and southwestern rice district, all there is generation in each large producing region, and Yunnan Province, with regard to You105Ge county, this disease occurs.This disease is comparatively large to yield effect, and output minimizing reaches 20%-30%, and can reach 50%-60% time serious, even No kernels or seeds are gathered, as in a year of scarcity.General long-grained nonglutinous rice injury overweights round-grained rice glutinous rice, and late rice injury overweights early rice.Its host, except paddy rice, also comprises dryland rice, wild-rice, and the white and Leersia Sw of water chestnut is natural host plant, but it is very uncommon that these plants fall ill.In addition also have water chestnut sheath chaff that is white and Leersia careless, batch shell grass, Semen Euphorbiae and maggot grass etc.
At present, bacterial blight of rice from 1884 since Japan, in succession in Asia, Europe, Oceania, Africa and North America be all found.The own extend over the entire globe rice cropping area of its occurrence scope, just there is Japan in the country of morbidity on the books in countries in the world, the U.S., China grade for 27 countries are wherein with Japan, and India and China fall ill comparatively serious.
Xanthomonas oryzae pv. oryzicola belongs to Prokaryota (Procaryotes), gracilicute door (Gracilicutes), the rice Xanthomonas campestris of Xanthomonas campestris section xanthomonas (Xanthomonas).Germ size is about 1.0-2.5 μm or 0.4-0.6 μm.Unit cell is paired once in a while, but does not become chain, does not form gemma and pod membrane, has the raw single flagellum of end, can move about, without spore, without pod membrane.Gramstaining reaction negative, growth optimum temperuture is 25 1 28 DEG C, and fatal temperature is 51 DEG C.On gravy peptone nutrient agar, growth of pathogenic bacteria is fast, and bacterium colony is often in faint yellow, circular, smooth, and regular edges is dome-shaped, thick, aerobic.Though the pathogenic and phene of this germ and rice leaf spot bacteria has a great difference, its inherited character and Physiological-biochemical Characters have again very large similarity, therefore this bacterium should as the mutation of in bacterial blight of rice bacterial classification.
Bacterial stripe, cecospora spot sick also known as slice.It is the important quarantine seed-borne disease of a kind of paddy rice caused by Xanthomonas campestris PV.oryzicola (Xanthomonasoryzae pv.oryzicola).Mainly cause harm rice leaf and children age blade.All can occur from seedling stage to heading stage, but fall ill for many early stage to heading with tillering phase.And this germ not only can be attached to seed-coat sometimes also can be deposited with Interior Seed.Scab is confined to parenchyma cell between vein, be just the translucent point of deep green water soaking mode, expands up and down between vein very soon, becomes faint yellow slat spot, not wide owing to limiting scab by vein, under a few cases germ to cross vein extending transversely.On disease-resistant variety, scab is very short, and size is about 1x10mm, and in susceptible variety, scab is longer, sometimes can reach 30-50mm. or longer, and many scabs can be linked to be the withered spot of bulk.Scab two ends are that infiltrative type is green.Often on scab overflow the yellow bacterial ooze of a large amount of beading, jelly granule after dry.On bacterial leaf spot scab, bacterium is overflow and seldom seldom sees, and bacterial stripe is then often covered with little pearl bacterium liquid.Except paddy rice, also have the white and many wild-rices of Australia rice, broad-leaved rice, water chestnut etc. all can be infected and fall ill, as Oryza latifolia, Oryza brachyantha, Oryza breviligulate etc.On the late hybrid rice of the Yangtze valley, also there is big area to occur in recent years.The hazardness of bacterial leaf streak of rice to long-grained nonglutinous rice is maximum, has risen to the predominantly bacteria disease in south China, Central-South rice district.
At present, bacterial leaf streak of rice is mainly distributed in subtropical and tropical zones, as Philippines, Thailand, India etc., from Area distribution, mainly concentrates on Asia and Africa.Southern provinces and regions are mainly distributed in, as Guangdong, Guizhou, Yunnan, Sichuan, Zhejiang, Jiangxi, Hunan, Hubei etc. at China's bacterial leaf streak of rice.
2007, the Ministry of Agriculture of China promulgated " People's Republic of China (PRC) enter the territory Plant Quarantine harmful organism register ", discloses and comprises harmful insect, mollusk, fungi, prokaryotic organism, nematode and virus etc., totally 435 kinds of harmful organisms.Wherein infect seed-borne disease mainly xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola), the rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) etc. of rice crop.These two kinds of germs not only can be infected rice crop and can be carried out long-distance communications by infected seed. and borrow the cutter, vessel etc. of wind and rain, irrigation water and entomochory and farming operation to carry out closely. and cause agricultural output to decline, hinder China's agricultural development greatly.
At present, observation of symptoms carries out pathogenic to detect a kind of conventional method, and observation of symptoms method is injured the symptom feature at position to reach the object of detection by direct making plant.Pathology types mainly contains: wilt, spot ulcer, soft rot, deformity and scab etc.Emphatically it is to be noted that the comparison of symptom characteristic between the position of morbidity, the scope of symptom variation and different onset individuality during observation of symptoms, but some disease pathogeneticing characteristics are but extremely similar, only be difficult to distinguish with naked eyes, also being difficult to distinguish when the same plant of two kinds or more disease infestation. such as xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria are only difficult to distinguish with naked eyes, only are more difficult to distinguish with observation of symptoms when Combined Infection.
Microscopic examination method cuts fritter diseased tissues at low power Microscopic observation, as can be observed thalline and spray bacterium phenomenon, then can be judged as Micobial Disease.In addition, utilize the microscopic structure of different bacterium different, and differently from the avidity of all kinds of dyestuff also can to pick out accurately by the bacterial classification infected.But for the different germs that microscopic structure is similar, microscopic examination method is just difficult to the kind distinguishing germ, as xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria.
Utilize PCR (polymerase chain reaction, Polymerase Chain Reaction is called for short PCR) method to detect crop cause of disease and have the advantages such as sensitive, quick, high specificity, its specificity and susceptibility are then better than observation of symptoms and isolation cultivation method.
Chinese Patent Application No. be 03105358.0 " being applied to probe and primer sequence that rice leaf spot bacteria detects " (its publication number is CN1524964, and publication date is on 09 01st, 2004) utilize probe technique to accept subbase because of sequence difference according to siderophore to devise Auele Specific Primer and probe.This probe is utilized to detect carry disease germs rice paddy seed and blade.This probe can directly fast be identified bacterial leaf spot pathogenic bacteria, and without the need to being separated germ and nucleic acid extraction from rice paddy seed, directly with seed leach liquor for template detects.Utilize this probe in detecting high specificity, can Direct Identification to subspecies.Chinese Patent Application No. be 201110035412.5 " for detecting the primer of rice leaf spot bacteria, method and test kit " (its publication number is CN102618628A, publication date is on 09 01st, 2004) devise a kind of primer for detecting rice leaf spot bacteria bacterial strain GD414, and optimize reaction system, can easy, promptly GD414 and other bacterial leaf-blight bacteria strain are made a distinction.But above-mentioned patent can only single detection rice leaf spot bacteria, can not accomplish the Multiple detection of xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria.
Chinese Patent Application No. is the " rice leaf spot bacteria of 201010218670.2, xanthomonas oryzae pv. oryzicola, the detection method of citrus ulcer bacteria and cabbage black rot bacterium " (its publication number is CN101921843A, publication date is on December 22nd, 2010) carry out PCR and react amplification and obtain PCR primer detecting the DNA profiling of sample by two pairs of primers simultaneously, then specific probe hybridization PCR primer and gene chip arranged, according to the results of hybridization positive whether, judge whether to detect sample containing rice leaf spot bacteria, xanthomonas oryzae pv. oryzicola, citrus ulcer bacteria or cabbage black rot bacterium, this patent disposablely can complete detection to 4 kinds of bacteriums, and detected result is simple and clear, easily judges.But the detection method that above-mentioned patent builds is carrying out the DNA profiling detecting sample also needing the specific probe that PCR primer and gene chip are arranged to hybridize after PCR reaction amplification obtains PCR primer, the result detected is determined, the time-consuming effort of testing process and high expensive according to results of hybridization.Can not accomplish to detect fast.
Chinese Patent Application No. be 200910030127.7 " the padlock probe of rice leaf spot bacteria and X. c. pv. oryzicola bacterium and multiple detection method " (its publication number is CN101608235, publication date is on December 23rd, 2009) respectively devise a padlock probe for rice leaf spot bacteria and X. c. pv. oryzicola bacterium, and rice leaf spot bacteria and Xanthomonas campestris PV.oryzicola can be detected in conjunction with Macroaary technology simultaneously, the specificity that tool is stronger, susceptibility and stability, for the detection of rice leaf spot bacteria and Xanthomonas campestris PV.oryzicola provides fast, sensitive, special technological method.Its Cleaning Principle of detection method that above-mentioned patent builds is first with desmoenzyme, the padlock probe of " unblanking " state head and the tail to be combined with DNA profiling, the padlock probe be not combined completely with DNA profiling is eliminated again with ligase enzyme, and make to connect into ring-type with DNA profiling in conjunction with right-on " unblanking " state padlock probe head and the tail, finally with the padlock probe of large fragment amplification enzymatic amplification ring-type.Complete reaction process.Detected through gel electrophoresis or macroarray finally can be adopted to carry out hybridization check.Testing process is loaded down with trivial details consuming time, and testing cost is higher.That can not accomplish to detect is quick, operation simple.
Summary of the invention
Xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola), rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) are all the seed-borne diseases mainly infecting rice crop, often infect same plant simultaneously.The object of the invention is the existing technological operation for detecting these two kinds of cause of diseases simultaneously of solution loaded down with trivial details, take time and effort, the technical problem that cost is higher, there is provided a kind of accurate for these two kinds of cause of diseases energy, fast, simply, lower-cost multiple PCR detection kit, multi-PCR detection method and primer special thereof.
Technical scheme of the present invention is as follows:
1. the primer special X-b of a rapid detection rice leaf spot bacteria, it is characterized in that: the primer special X-b of described detection rice leaf spot bacteria is made up of upstream primer and downstream primer, the base sequence of the upstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:1, the base sequence of the downstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:2, and target product fragment length is 461bp.
2. the primer special X-t of a rapid detection xanthomonas oryzae pv. oryzicola, it is characterized in that: the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is made up of upstream primer and downstream primer, the base sequence of the upstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQ ID NO:3, the base sequence of the downstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQID NO:4, and target product fragment length is 348bp.
3. for while rapid detection xanthomonas oryzae pv. oryzicola and the test kit of rice leaf spot bacteria, it is characterized in that described test kit comprises following optimum multi-PRC reaction system and optimum reaction condition:
Described optimum multi-PRC reaction system is: 10 × PCR reaction buffer 2.5 μ l, the MgCl of 25mM
2the dNTP Mixture 2 μ l of 1.5 μ l, 2.5mM; The each 0.5 μ l of upstream and downstream primer of the primer special X-b of 1 μM of detection rice leaf spot bacteria, the Taq polymerase 0.5 μ l of each 0.5 μ l, the 5U/ μ l of upstream and downstream primer of the primer special X-t of 1 μM of detection xanthomonas oryzae pv. oryzicola, 1 × 10
5cfu/mL ~ 1 × 10
8rice paddy seed sample 0.5 μ l, the RNase Free dH to be checked of cfu/mL
2o complements to 25 μ l;
The base sequence of the upstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ IDNO:1, the base sequence of the downstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:2, and target product fragment length is 461bp;
The base sequence of the upstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQID NO:3, the base sequence of the downstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQ ID NO:4, and target product fragment length is 348bp;
Described optimum reaction condition is:
95 DEG C of denaturation 5min, from 95 DEG C of sex change 30sec, 56 DEG C of annealing 60sec, 72 DEG C extend 60sec and run 30 circulations, and 72 DEG C extend 10min.
4. a molecular biology method for rapid detection xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria while, is characterized in that comprising the following steps:
(1) preparation of rice paddy seed sample to be checked
Rice paddy seed to be checked gets the phosphoric acid buffer that 20 add 5ml sterilizing, crush in the mortar of sterilizing and make extracting solution, 5h whirlpool mixing 5min is placed at 25 DEG C, the phosphoric acid buffer adding sterilizing is again diluted to the suspension of 100 times, get 100 microlitres and be placed in dull and stereotyped upper 28 DEG C of cultivation 50h, with the sterilized water process corresponding time for contrast; Lawn sterilized water on flat board is eluted, obtains 1 × 10
5cfu/mL-1 × 10
8the rice paddy seed sample to be checked of cfu/mL;
(2) multi-PRC reaction
Multi-PRC reaction system is: 10 × PCR reaction buffer 2.5 μ l, the MgCl of 25mM
2the dNTP Mixture 2 μ l of 1.5 μ l, 2.5mM; The each 0.5 μ l of upstream and downstream primer of the primer special X-b of 1 μM of detection rice leaf spot bacteria, the Taq polymerase 0.5 μ l of each 0.5 μ l, the 5U/ μ l of upstream and downstream primer of the primer special X-t of 1 μM of detection xanthomonas oryzae pv. oryzicola, 1 × 10
5cfu/mL-1 × 10
8rice paddy seed sample 0.5 μ l, the RNase Free dH to be checked of cfu/mL
2o complements to 25 μ l;
The base sequence of the upstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ IDNO:1, the base sequence of the downstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:2, and target product fragment length is 461bp;
The base sequence of the upstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQID NO:3, the base sequence of the downstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQ ID NO:4, and target product fragment length is 348bp;
Multi-PRC reaction condition is:
95 DEG C of denaturation 5min, from 95 DEG C of sex change 30sec, 56 DEG C of annealing 60sec, 72 DEG C extend 60sec and run 30 circulations, and 72 DEG C extend 10min;
(3) electrophoresis detection and judgement
Multi-PRC reaction stop after, get 5 μ l PCR primer, with 1.5% agarose electrophoresis detection amplification, if it is consistent with the target product fragment length of prediction to obtain clip size, then in this rice paddy seed sample to be checked with corresponding bacterium.
Compared with prior art, the invention has the beneficial effects as follows:
Multiple PCR detection kit of the present invention, multi-PCR detection method, by the selection of the target fragment to standard bacteria rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, the design of two pairs of primer specials, annealing temperature and time, elongating temperature and time, the parameter designing such as the consumption of each reacted constituent, when making the present invention detect this two kinds of germs at the same time, specificity is strong especially, check result is accurate, inspection operation simplifies, only need judge that whether PCR primer clip size and the target product fragment length of prediction be consistent can draw detected result accurately immediately, without the need to cloning again, order-checking and sequence alignment, overcome prior art also to need the specific probe that PCR primer and gene chip are arranged to hybridize, testing process not only time-consuming effort but also high expensive, or need in prior art first with ligase enzyme, the padlock probe of " unblanking " state head and the tail to be combined with DNA profiling, the padlock probe be not combined completely with DNA profiling is eliminated again with excision enzyme, and make to connect into ring-type with DNA profiling in conjunction with right-on " unblanking " state padlock probe head and the tail, finally with the padlock probe of large fragment amplification enzymatic amplification ring-type, complete reaction process, detected through gel electrophoresis or macroarray finally can be adopted to carry out hybridization check, its testing process is loaded down with trivial details consuming time, and the defect that testing cost is higher.
Above-mentioned prior art at least needs 4-8 hour, and synthesis two padlock probes at least need 1600 yuan and need in testing process to use expensive gene chip or be of little use and more expensive excision enzyme or large fragment amplification enzyme.Adopt the inventive method only to need 2 hours can obtain detected result, the synthesis of four primers only needs 80 yuan and only needs common pcr amplification reagent to complete detection, without the need to the expensive experiment consumptive material such as padlock probe of costliness, reduces testing cost.Therefore, the present invention proposes new technical scheme, when its method, test kit, primer special detect these two kinds of germs simultaneously, not only specificity is strong especially, and fast, accurately, judge easy, schedule of operation simple, without the need to carrying out cloning again, checking order and sequence alignment can directly judge, testing cost is lower.
The present invention is all significant for the bacteria-treating of the academic research of these two kinds of germs and commute infection plant simultaneously.
In sequence table shown in SEQ ID NO:1 is the base sequence that the present invention detects the upstream primer of the primer special X-b of rice leaf spot bacteria.
In sequence table shown in SEQ ID NO:2 is the base sequence that the present invention detects the downstream primer of the primer special X-b of rice leaf spot bacteria.
In sequence table shown in SEQ ID NO:3 is the base sequence that the present invention detects the upstream primer of the primer special X-t of xanthomonas oryzae pv. oryzicola.
In sequence table shown in SEQ ID NO:4 is the base sequence that the present invention detects the downstream primer of the primer special X-t of xanthomonas oryzae pv. oryzicola.
Accompanying drawing explanation
Fig. 1 is that the primer special X-b of applying detection rice leaf spot bacteria carries out pcr amplification to standard bacteria rice leaf spot bacteria, to the PCR primer obtained through 1.5% agarose gel electrophoresis result figure.M is Marker, and swimming lane 1 is negative control, and swimming lane 2 is standard bacteria xanthomonas oryzae pv. oryzicola, and swimming lane 3 is standard bacteria rice leaf spot bacteria, and target stripe length is 461bp.
Fig. 2 is that the primer special X-t of applying detection xanthomonas oryzae pv. oryzicola carries out pcr amplification to standard bacteria xanthomonas oryzae pv. oryzicola, to the PCR primer obtained through 1.5% agarose gel electrophoresis result figure.M is Marker, and swimming lane 1 is negative control, and swimming lane 2 is standard bacteria rice leaf spot bacteria, and swimming lane 3 is standard bacteria xanthomonas oryzae pv. oryzicola, and target stripe length is 348bp.
Fig. 3 is the product 1.5% agarose gel electrophoresis result figure utilizing the mixing sample of primer special X-t to standard bacteria rice leaf spot bacteria, standard bacteria xanthomonas oryzae pv. oryzicola and these two kinds of standard bacteria of the primer special X-b of test kit of the present invention and rice leaf spot bacteria and detection xanthomonas oryzae pv. oryzicola to carry out multiplexed PCR amplification.In figure: M is that Marker, A and B represent the position that each fragment is horizontal in the drawings.Swimming lane 1 is standard bacteria xanthomonas oryzae pv. oryzicola, and swimming lane 2 is standard bacteria rice leaf spot bacteria, the mixed bacteria liquid sample of swimming lane 3 standard bacteria rice leaf spot bacteria and standard bacteria xanthomonas oryzae pv. oryzicola, and swimming lane 4 is negative control; In swimming lane 1, in B position and swimming lane 3, the band of B position is the target stripe of standard bacteria xanthomonas oryzae pv. oryzicola, and target stripe length is 348bp.In swimming lane 2, in A position and swimming lane 3, the band of A position is the target stripe of standard bacteria rice leaf spot bacteria, and target stripe length is 461bp.
Fig. 4 is that the primer special X-t of the primer special X-b and detection xanthomonas oryzae pv. oryzicola utilizing test kit of the present invention, detection method or detect rice leaf spot bacteria carries out multiplexed PCR amplification gained PCR primer 1.5% agarose gel electrophoresis result figure to the commercially available rice paddy seed sample being numbered 1 to 20.In figure: M is Marker, two bands of symbol+occur from top to bottom are followed successively by the positive control of standard bacteria rice leaf spot bacteria and standard bacteria xanthomonas oryzae pv. oryzicola, symbol-and be negative control.The commercially available rice paddy seed sample of swimming lane 1 to 20 for being numbered 1 to 20, the rice paddy seed sample of swimming lane 1 to swimming lane 20 is negative for detecting.
Fig. 5 is that the primer special X-t of the primer special X-b and detection xanthomonas oryzae pv. oryzicola utilizing test kit of the present invention, detection method or detect rice leaf spot bacteria carries out multiplexed PCR amplification gained PCR primer 1.5% agarose gel electrophoresis result figure to the commercially available rice paddy seed sample being numbered 21 to 39.In figure: M is Marker, two bands of symbol+occur from top to bottom are followed successively by the positive control of standard bacteria rice leaf spot bacteria and standard bacteria xanthomonas oryzae pv. oryzicola, symbol-and be negative control.The commercially available rice paddy seed sample of swimming lane 21 to 39 for being numbered 21 to 39, swimming lane 22 is the target stripe of xanthomonas oryzae pv. oryzicola, target stripe length is 348bp, and the rice paddy seed sample of swimming lane 21 is negative for detecting, and the rice paddy seed sample of swimming lane 23 to swimming lane 39 is negative for detecting.
Fig. 6 is that the primer special X-t of the primer special X-b and detection xanthomonas oryzae pv. oryzicola utilizing test kit of the present invention, detection method or detect rice leaf spot bacteria carries out multiplexed PCR amplification gained PCR primer 1.5% agarose gel electrophoresis result figure to the commercially available rice paddy seed sample being numbered 40 to 47.In figure: M is Marker, two bands of symbol+occur from top to bottom are followed successively by the positive control of standard bacteria rice leaf spot bacteria and standard bacteria xanthomonas oryzae pv. oryzicola, symbol-and be negative control.The commercially available rice paddy seed sample of swimming lane 40 to 47 for being numbered 40 to 47, the target stripe of swimming lane to be 45 be rice leaf spot bacteria, target stripe length is 461bp.Swimming lane 40 to swimming lane 44, the rice paddy seed sample of swimming lane 46 to swimming lane 47 is negative for detecting.
Embodiment
Below in conjunction with embodiment, the invention will be further described, is convenient to understand the present invention better, but do not limit the present invention.
Experimental technique in the following example, is ordinary method, agents useful for same, material, all can buy from biochemical reagents shop and Seed Market.Rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) used and xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola) are and purchase available standard bacteria from Institute of Micro-biology.
By aseptic beef extract-peptone nutrient solution, (formula is extractum carnis 0.5g respectively for rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) in following examples and xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola) two kinds of bacteriums, peptone 1g, sodium-chlor 0.5g, distilled water 100mL, pH 7.0 ~ 7.2) 28 DEG C of constant-temperature table 180r/min cultivate 48h, and making concentration is respectively 1 × 10
8the bacterium liquid of the rice leaf spot bacteria of cfu/mL and concentration are 1 × 10
8the xanthomonas oryzae pv. oryzicola of cfu/mL.
The primer special X-b of embodiment 1 rapid detection rice leaf spot bacteria of the present invention and design thereof and use this to detect the conventional molecular biological method of rice leaf spot bacteria to primer special X-b.
(1) Design and synthesis of the primer special X-b of rice leaf spot bacteria is detected
The primer special X-b detecting rice leaf spot bacteria is according to hrp gene cluster 1 and 2 region gene fragment in rice leaf spot bacteria chromogene group, homology analysis is carried out with DNAMAN6.0, determine high conservative region, again with the Auele Specific Primer of Primer 6.0 primer-design software design, be numbered primer special X-b, the primer special X-b detecting rice leaf spot bacteria is made up of upstream primer and downstream primer, the base sequence of the upstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ IDNO:1, the base sequence of the downstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:2, target product fragment length is 461bp.
Described primer special X-b is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
(2) detect the molecular biology method of rice leaf spot bacteria with the primer special X-b detecting rice leaf spot bacteria, comprise the following steps:
1. the primer special X-b of the detection rice leaf spot bacteria described in above-mentioned steps (1) is synthesized.The primer special X-b of this detection rice leaf spot bacteria is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
2. the substance PCR of rice leaf spot bacteria reacts
With the bacterium liquid of rice leaf spot bacteria for template, the bacterium liquid of xanthomonas oryzae pv. oryzicola is positive control, blank nutrient solution after sterilizing makes negative control, by common PCR reaction system described in table 1 and PCR reaction conditions be: 95 DEG C of denaturation 5min, from 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s and carry out 35 circulations, and 72 DEG C extend 10min eventually and carry out pcr amplification.
Table 1 common PCR reaction system
In table 1: the upstream primer of primer special X-b is writing a Chinese character in simplified form of the upstream primer of the primer special X-b detecting rice leaf spot bacteria; The downstream primer of primer special X-b is writing a Chinese character in simplified form of the downstream primer of the primer special X-b detecting rice leaf spot bacteria.
5. electrophoresis detection
After PCR reaction terminating, get 5 μ l PCR primer, 1.5% agarose electrophoresis detects amplification, and the fragment that can amplify about 461bp is rice leaf spot bacteria, as shown in Figure 1.
6. Cloning and sequencing
(Qiagen Inc. is purchased from QIAquick PCR purification kit, Chatsworth, CA) purifying pcr amplification product, and (be purchased from Promega, Madison, WI with pGEM-T carrier, USA) connect, transformed by Escherichiacoli cell (JM-109), use standard biological schedule of operation, checked order by the biological company limited in Tyke, Beijing hundred AB13370DNA automatic sequencer.
7. sequence alignment
The sequence obtained is passed through NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) compare of analysis, and the consistence of the rice leaf spot bacteria hrp genecluster 1 and 2 region gene of (accession number is AB045311.1) reported with Genbank is 99%.Prove that the primer special X-b of this detection rice leaf spot bacteria has good specificity for rice leaf spot bacteria.
The primer special X-t of embodiment 2 rapid detection xanthomonas oryzae pv. oryzicola of the present invention and design thereof and use this to detect the conventional molecular biological method of xanthomonas oryzae pv. oryzicola to primer special X-t.
(1) Design and synthesis of the primer special X-t of xanthomonas oryzae pv. oryzicola is detected
The primer special X-t detecting xanthomonas oryzae pv. oryzicola is according to AvrRxo1-ORF1 gene fragment in xanthomonas oryzae pv. oryzicola chromogene group, homology analysis is carried out with DNAMAN6.0, determine high conservative region, again with the Auele Specific Primer of Primer 6.0 primer-design software design, be numbered primer special X-t, the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is made up of upstream primer and downstream primer, the base sequence of the upstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQ ID NO:3, the base sequence of the downstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQ ID NO:4, the target product fragment length of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is 348bp.Described primer special X-t is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
(1) detect the molecular biology method of xanthomonas oryzae pv. oryzicola with the primer special X-t detecting xanthomonas oryzae pv. oryzicola, comprise the following steps:
1. the primer special X-t of the detection xanthomonas oryzae pv. oryzicola described in above-mentioned steps (1) is synthesized.The primer special X-t of this detection xanthomonas oryzae pv. oryzicola is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
2. the substance PCR of xanthomonas oryzae pv. oryzicola reacts
With the bacterium liquid of xanthomonas oryzae pv. oryzicola for template, the bacterium liquid of rice leaf spot bacteria is positive control, blank nutrient solution after sterilizing makes negative control, by common PCR reaction system described in table 2 and PCR reaction conditions be: 95 DEG C of denaturation 5min, from 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s and run 35 circulations, and 72 DEG C extend 10min eventually and carry out pcr amplification.
Table 2 common PCR reaction system
In table 2: the upstream primer of primer special X-t is writing a Chinese character in simplified form of the upstream primer of the primer special X-t detecting xanthomonas oryzae pv. oryzicola; The downstream primer of primer special X-t is writing a Chinese character in simplified form of the downstream primer of the primer special X-t detecting xanthomonas oryzae pv. oryzicola.
5. electrophoresis detection
After PCR reaction terminating, get 5 μ l PCR primer, 1.5% agarose electrophoresis detects amplification.The fragment that can amplify about 348bp is xanthomonas oryzae pv. oryzicola, as shown in Figure 2.
6. Cloning and sequencing
(Qiagen Inc. is purchased from QIAquick PCR purification kit, Chatsworth, CA) purifying pcr amplification product, (Promega is purchased from, Madison, WI with pGEM-T carrier, USA) connect, and transformed by Escherichiacoli cell (JM-109), use standard biological schedule of operation, checked order by the biological company limited in Tyke, Beijing hundred AB13370DNA automatic sequencer.
7. sequence alignment
The sequence obtained is passed through NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) compare of analysis, and the consistence of (accession number is AY395713.1) the xanthomonas oryzae pv. oryzicola AvrRxo1-ORF1 gene reported with Genbank is 99%.Prove that the primer special X-t of this detection xanthomonas oryzae pv. oryzicola has good specificity for paddy rice xanthomonas oryzae pv. oryzicola.
Embodiment 3 test kit for quick and precisely detecting xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola) and rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) simultaneously provided by the invention comprises:
10 × PCR reaction buffer,
The MgCl of 25mM
2,
The dNTP Mixture of 2.5mM,
The upstream primer of the primer special X-b of 1 μM of detection rice leaf spot bacteria,
The downstream primer of the primer special X-b of 1 μM of detection rice leaf spot bacteria,
The upstream primer of the primer special X-t of 1 μM of detection xanthomonas oryzae pv. oryzicola,
The downstream primer of the primer special X-t of 1 μM of detection xanthomonas oryzae pv. oryzicola,
The Taq polymerase of 5U/ μ l,
RNase Free dH
2O；
Specification sheets is furnished with in described test kit, the optimum multi-PRC reaction system and optimum reaction condition of recommending with the present invention is recorded in specification sheets, described optimum multi-PRC reaction system is as shown in table 3, optimum reaction condition is: 95 DEG C of denaturation 5min, from 95 DEG C of sex change 30sec, 56 DEG C of annealing 60sec, 72 DEG C extend 60sec and run 30 circulations; 72 DEG C extend 10min.
The optimum multi-PRC reaction system of table 3 invention
In table 3:
The upstream primer of primer special X-b is writing a Chinese character in simplified form of the upstream primer of the primer special X-b detecting rice leaf spot bacteria;
The downstream primer of primer special X-b is writing a Chinese character in simplified form of the downstream primer of the primer special X-b detecting rice leaf spot bacteria;
The upstream primer of primer special X-t is writing a Chinese character in simplified form of the upstream primer of the primer special X-t detecting xanthomonas oryzae pv. oryzicola;
The downstream primer of primer special X-t is writing a Chinese character in simplified form of the downstream primer of the primer special X-t detecting xanthomonas oryzae pv. oryzicola;
The base sequence of the upstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ IDNO:1, the base sequence of the downstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:2, and target product fragment length is 461bp;
The base sequence of the upstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQID NO:3, the base sequence of the downstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQ ID NO:4, and target product fragment length is 348bp;
Embodiment 4 by multiple PCR detection kit of the present invention and detection method respectively to the detection of the mixing sample of rice leaf spot bacteria, xanthomonas oryzae pv. oryzicola, rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola
1. respectively with the bacterium liquid of rice leaf spot bacteria, xanthomonas oryzae pv. oryzicola, rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola biased sample for template, make negative control with the blank beef extract-peptone nutrient solution after sterilizing as nutrient solution.The optimum multi-PRC reaction system described in table 3 (now rice paddy seed sample to be checked is respectively the bacterium liquid balanced mix sample of the bacterium liquid sample of rice leaf spot bacteria, the bacterium liquid sample of xanthomonas oryzae pv. oryzicola, rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola) and reaction conditions is used to be respectively: 95 DEG C of denaturation 5min; From 94 DEG C of sex change 30sec, 56 DEG C of annealing 60sec, 72 DEG C extend 60sec and run 30 circulations; 72 DEG C extend 10min and carry out multiplexed PCR amplification.
2. after PCR reaction terminating, get 5 μ l PCR primer, detect amplification by 1.5% agarose electrophoresis.About 461bp can be amplified respectively, the fragment that about 348bp and about 461bp and about 348bp combine can think successfully detect rice leaf spot bacteria, xanthomonas oryzae pv. oryzicola, rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola mixing sample as shown in Figure 3.From Fig. 3, swimming lane 1 very clearly can see that length is the band of 348bp, swimming lane 2 very clearly can see that length is the band of 461bp, swimming lane 3 very clearly can see that length is the band of 348bp and 461bp, and the negative control of swimming lane 4 is without band, illustrate: with multiple PCR detection kit of the present invention and multi-PCR detection method respectively to the accuracy that the mixing sample of standard bacteria rice leaf spot bacteria, xanthomonas oryzae pv. oryzicola, rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola detects.
Embodiment 5 multiple PCR detection kit of the present invention and multi-PCR detection method detect the Carriage (each rice paddy seed all by the following method) of 47 kinds of commercially available rice paddy seeds
(1) preparation of rice paddy seed sample to be checked
Each rice paddy seed to be checked gets the phosphoric acid buffer that 20 add 5ml sterilizing, thoroughly crush in the mortar of sterilizing and make extracting solution, 5h whirlpool mixing 5min is placed at 25 DEG C, the phosphoric acid buffer adding sterilizing is again diluted to the suspension of 100 times, get 100 microlitres and be placed in dull and stereotyped upper 28 DEG C of cultivation 50h, with the sterilized water process corresponding time for contrast.Lawn sterilized water on flat board is eluted, obtains 1 × 10
5cfu/mL-1 × 10
8the rice paddy seed sample to be checked of cfu/mL; Described phosphoric acid buffer liquid and preparation method thereof: NaCl 80g, Na
2hPO
42H
2o 32.3g, NaH
2pO
42H
2o 4.5g, adds distilled water 885mL, and adjust pH to 7.2 with HCl/NaOH, last constant volume is 1000mL.
(2) multi-PRC reaction
Get rice paddy seed sample 0.5 μ l to be checked described in step (1) in PCR reaction tubes, add 10 × PCR reaction buffer 2.5 μ l respectively, the MgCl of 25mM
2the dNTP Mixture 2 μ l of 1.5 μ l, 2.5mM; The each 0.5 μ l of upstream and downstream primer of the primer special X-b of 1 μM of detection rice leaf spot bacteria, the each 0.5 μ l of upstream and downstream primer of the primer special X-t of 1 μM of detection xanthomonas oryzae pv. oryzicola, the Taqpolymerase 0.5 μ l of 5U/ μ l, RNase Free dH
2o complements to 25 μ l; The base sequence of the upstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:1, the base sequence of the downstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:2, and target product fragment length is 461bp; The base sequence of the upstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQ ID NO:3, the base sequence of the downstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQ ID NO:4, and target product fragment length is 348bp.
Reaction conditions is: 95 DEG C of denaturation 5min, from 94 DEG C of sex change 30sec, and 56 DEG C of annealing 60sec, 72 DEG C extend 60sec and run 30 circulations; 72 DEG C extend 10min.
(3) electrophoresis detection and judgement
Multi-PRC reaction stop after, get 5 μ l PCR primer, with 1.5% agarose electrophoresis detection amplification, if it is consistent with the target product fragment length of prediction to obtain clip size, then in this seed sample to be checked with corresponding bacterium; Not on the same day in triplicate.
Detected result is as Fig. 4, Fig. 5, Fig. 6: do not come to the same thing in triplicate on the same day.
Experimental result shows: in the swimming lane representing 47 kinds of rice paddy seed samples, and swimming lane 22 detected the target stripe 348bp of xanthomonas oryzae pv. oryzicola, and swimming lane 45 detected the target stripe 461bp of rice leaf spot bacteria, and other swimming lanes are negative for detecting.Do not come to the same thing in triplicate on the same day.This illustrates in the 47 kinds of rice paddy seeds detected, and corresponding No. 22 seeds are with xanthomonas oryzae pv. oryzicola, and No. 45 seeds are with rice leaf spot bacteria.Show: multiple PCR detection kit of the present invention and multi-PCR detection method of the present invention do not need to clone again, order-checking and sequence alignment, obtain clip size consistent with target of prediction fragment, just can determine in this sample with corresponding bacterium, its specificity simultaneously detecting rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) and xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola) two kinds of germs is strong especially, detected result is accurate, fast, only need can obtain detected result in 2 hours, trace routine is easy, reduce testing cost.
<110> Yunnan Prov Agriculture University
Yunnan Lin Peng agricultural science and technology company limited
The multiple PCR detection kit of two kinds of seed-borne diseases and primer special thereof and multi-PCR detection method on <120> paddy rice
<130> /
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> rice leaf spot bacteria (Xanthomonas oryzae pv. Oryzae)
<400> 1
attcgttcct gtaaccgtga 20
<210> 2
<211> 20
<212> DNA
<213> rice leaf spot bacteria (Xanthomonas oryzae pv. Oryzae)
<400> 2
cacatgctgg ttcttacgtt 20
<210> 3
<211> 18
<212> DNA
<213> xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola)
<400> 3
gccgaccatg acttaccg 18
<210> 4
<211> 19
<212> DNA
<213> xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola)
<400> 4
tccgtaccct gtagtctcg 19
Claims (4)
1. the primer special X-b of a rapid detection rice leaf spot bacteria, it is characterized in that: the primer special X-b of described detection rice leaf spot bacteria is made up of upstream primer and downstream primer, the base sequence of the upstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:1, the base sequence of the downstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:2, and target product fragment length is 461bp.
2. the primer special X-t of a rapid detection xanthomonas oryzae pv. oryzicola, it is characterized in that: the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is made up of upstream primer and downstream primer, the base sequence of the upstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQ ID NO:3, the base sequence of the downstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQID NO:4, and target product fragment length is 348bp.
3. for while rapid detection xanthomonas oryzae pv. oryzicola and the test kit of rice leaf spot bacteria, it is characterized in that described test kit comprises following optimum multi-PRC reaction system and optimum reaction condition:
Described optimum multi-PRC reaction system is: 10 × PCR reaction buffer 2.5 μ l, the MgCl of 25mM
2the dNTP Mixture 2 μ l of 1.5 μ l, 2.5mM; The each 0.5 μ l of upstream and downstream primer of the primer special X-b of 1 μM of detection rice leaf spot bacteria, the Taq polymerase 0.5 μ l of each 0.5 μ l, the 5U/ μ l of upstream and downstream primer of the primer special X-t of 1 μM of detection xanthomonas oryzae pv. oryzicola, 1 × 10
5cfu/mL ~ 1 × 10
8rice paddy seed sample 0.5 μ l, the RNase Free dH to be checked of cfu/mL
2o complements to 25 μ l;
The base sequence of the upstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ IDNO:1, the base sequence of the downstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:2, and target product fragment length is 461bp;
The base sequence of the upstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQID NO:3, the base sequence of the downstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQ ID NO:4, and target product fragment length is 348bp;
Described optimum reaction condition is:
95 DEG C of denaturation 5min, from 95 DEG C of sex change 30sec, 56 DEG C of annealing 60sec, 72 DEG C extend 60sec and run 30 circulations, and 72 DEG C extend 10min.
4. a molecular biology method for rapid detection xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria while, is characterized in that comprising the following steps:
(1) preparation of rice paddy seed sample to be checked
Rice paddy seed to be checked gets the phosphoric acid buffer that 20 add 5ml sterilizing, crush in the mortar of sterilizing and make extracting solution, 5h whirlpool mixing 5min is placed at 25 DEG C, the phosphoric acid buffer adding sterilizing is again diluted to the suspension of 100 times, get 100 microlitres and be placed in dull and stereotyped upper 28 DEG C of cultivation 50h, with the sterilized water process corresponding time for contrast; Lawn sterilized water on flat board is eluted, obtains 1 × 10
5cfu/mL-1 × 10
8the rice paddy seed sample to be checked of cfu/mL;
(2) multi-PRC reaction
Multi-PRC reaction system is: 10 × PCR reaction buffer 2.5 μ l, the MgCl of 25mM
2the dNTP Mixture 2 μ l of 1.5 μ l, 2.5mM; The each 0.5 μ l of upstream and downstream primer of the primer special X-b of 1 μM of detection rice leaf spot bacteria, the Taq polymerase 0.5 μ l of each 0.5 μ l, the 5U/ μ l of upstream and downstream primer of the primer special X-t of 1 μM of detection xanthomonas oryzae pv. oryzicola, 1 × 10
5cfu/mL-1 × 10
8rice paddy seed sample 0.5 μ l, the RNase Free dH to be checked of cfu/mL
2o complements to 25 μ l;
The base sequence of the upstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ IDNO:1, the base sequence of the downstream primer of the primer special X-b of described detection rice leaf spot bacteria is as shown in SEQ ID NO:2, and target product fragment length is 461bp;
The base sequence of the upstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQID NO:3, the base sequence of the downstream primer of the primer special X-t of described detection xanthomonas oryzae pv. oryzicola is as shown in SEQ ID NO:4, and target product fragment length is 348bp;
Multi-PRC reaction condition is:
95 DEG C of denaturation 5min, from 95 DEG C of sex change 30sec, 56 DEG C of annealing 60sec, 72 DEG C extend 60sec and run 30 circulations, and 72 DEG C extend 10min;
(3) electrophoresis detection and judgement
Multi-PRC reaction stop after, get 5 μ l PCR primer, with 1.5% agarose electrophoresis detection amplification, if it is consistent with the target product fragment length of prediction to obtain clip size, then in this rice paddy seed sample to be checked with corresponding bacterium.
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| CN109735512A (en) * | 2019-03-18 | 2019-05-10 | 华中农业大学 | Corn gene ZmACO2 is improving the application in corn yield |
| CN114350758A (en) * | 2021-12-31 | 2022-04-15 | 杭州飞时达生物科技有限公司 | Primer group and kit for rapidly detecting pathogenic bacteria of bacterial leaf blight of rice based on LAMP |
| CN115725753A (en) * | 2022-08-05 | 2023-03-03 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Dual PCR detection primer for two rice bacterial diseases and application thereof |
| CN116904620A (en) * | 2023-03-23 | 2023-10-20 | 三亚中国检科院生物安全中心 | Primer group, kit and method for rapidly detecting rice pathogenic bacteria based on MNP |
| CN117535436A (en) * | 2024-01-05 | 2024-02-09 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting rice bacterial leaf spot bacteria based on CRISPR/Cas12a-RPA and application |
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