CN104862404B - The multiple PCR detection kit of two kinds of seed-borne diseases and its primer special and multi-PCR detection method on rice - Google Patents
The multiple PCR detection kit of two kinds of seed-borne diseases and its primer special and multi-PCR detection method on rice Download PDFInfo
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Abstract
The present invention discloses on a kind of rice the multiple PCR detection kit of two kinds of seed-borne diseases and its primer special and multi-PCR detection method.Two kinds of seed-borne diseases of the present invention are bacterial blight of rice and bacterial leaf streak of rice.Kit and method of the present invention are equipped with the primer special X b of a pair of of detection rice leaf spot bacteria, and target product fragment length is 461bp.The primer special X t of a pair of quick detection xanthomonas oryzae pv. oryzicola, target product fragment length is 348bp, optimal multi-PRC reaction system and reaction condition when detecting two kinds of sick cause of diseases at the same time.The present invention detects the detection program, easy to operate of two kinds of cause of diseases simultaneously, and quick and precisely, cost is relatively low, without being cloned, being sequenced again and sequence alignment.
Description
Technical field
The present invention relates to the technical field of molecular biological detection of two kinds of pathogenics, and in particular to bacterial blight of rice
Bacterium (Xanthomonas oryzae pv.oryzae) and Xanthomonas campestris PV.oryzicola (Xanthomonas oryzae
Pv.oryzicola multiple molecular biological detection method).
Background technology
China is the fourth-largest importer of Agricultural Products Trade and the fifth-largest exported country, makes China under the trend of economic globalization
The further expansion of foreign trade, the allocation and transportation of plants and plant product are increased, while also bring more serious harmful life
The invasion of object threatens, this oneself through become endanger one of China's bio-diversity, ecological environment and national economy it is particularly significant and
The problem of urgent.Nowadays, the rice paddy seed industrial market in China is compared to the main Rice Production state such as Southeast Asia and South America
It compares, advantage is more apparent, no matter variety classification or the methods of conducting trade all gradually tend to variation.But due to the drive of economic interests
Dynamic, some illegal retailers produce privately, manage rice paddy seed.This not only serious quality and letter for compromising China's seed export
Reputation, also results in of poor quality, and the seed with malicious or unsuitable ecological region planting condition comes into the market.Each entry and exit port and the place of production are planted
Object disease inspection and quarantine an urgent demand efficient and sensible, fast and simple diagnostic techniques and instrument, to strengthen port quarantine, are constructed anti-
First of barrier of the external epidemic disease evil invasion of model, to ensureing China's agricultural production security, serves the outlet of agricultural product and state's internal trade
Easily it is of great immediate significance.
Two kinds of seed-borne diseases refer to respectively by rice leaf spot bacteria (Xanthomonas on rice of the present invention
Oryzae pv.oryzae) caused by bacterial blight of rice and by xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae
Pv.oryzicola bacterial leaf streak of rice caused by).
Bacterial blight of rice is one as caused by rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae)
The important quarantine seed-borne disease of kind rice.Its thalline is in rod-short, and unit cell, single flagellum, extremely raw or sub- extremely raw, no bud is embraced, nothing
Capsule, the exocellular polysaccharide that thalline has cement outside surround, and Gram-negative is aerobic.In gravy peptone agar medium culture
Bacterium colony honey gold or faint yellow on base, circular, periphery is neat, homogeneous, surface elevation, and along shinny, viscosity generates non-aqueous light
The uranidin of dissolubility.Pathogen growth optimum temperature be 25-30 DEG C, minimum 5 DEG C, 40 DEG C of highest, adhesive-free film protection under 53 DEG C 10
It is minute lethal, have glued membrane protection it is lower 57 DEG C 10 minutes it is lethal.Germ optimum acid-base value is pH6.5-7.0.
This kind of germ be also known as white leaf pest, burn, cogongrass pest etc..The entire breeding time of rice can be killed, especially seedling stage,
Point ear period is aggrieved most heavy, and each organ can catch an illness, and blade is most easily caught an illness.And the germ not only can be attached to the surface of the seed sometimes
Interior Seed can be also deposited with.Its symptom is common because germ infestation position, variety resistance, environmental condition have larger difference
Mainly there are the withered type of leaf, 3 type of acute wilting type, foxiness or brown stain type：
(1) the withered type of leaf, blade of mainly causing harm, leaf sheath of also causing harm when serious, morbidity first since blade tip or leaf margin, first go out
Existing dirty-green water soaking mode wire spot, forms yellow-white scab along wire spot quickly, then extends, becomes along leaf margin both sides or middle rib
Yellowish-brown, finally in withered white, scab edge boundary is apparent.
(2) acute wilting type, from seedling stage to tillering period, germ easily sends out when invading vascular bundle from root system or basal part of stem wound
Disease.And stem is fallen ill simultaneously, and lobus cardiacus dehydration green grass or young crops is withered, wilting death, and rest blade also successively blue or green withered curling, then complete stool is withered
Extremely, the situation that only lobus cardiacus is withered is also had.
(3) foxiness or brown stain type, more common on disease-resistant variety, germ is invaded by artificial leaf-cutting or wound, in temperature
Low or unfavorable onset condition when brown necrotic reaction band occurs in scab periphery, can stagnate extension.
Yellow type symptom is rarer, and it is not withered to be mainly manifested in early stage lobus cardiacus, and can generate irregular chlorisis spot,
Later stage of catching an illness can develop into withered and yellow spot.
The germ is as a kind of important quarantine seed-borne disease of rice, and harm is only second to bacterial leaf streak of rice, at me
In addition to Xinjiang, Tibet and southwestern rice region, there is generation in major producing region for state, and with regard to You105Ge counties this disease occurs for Yunnan Province.The disease is right
Yield effect is larger, and yield is reduced up to 20%-30%, up to 50%-60% or even No kernels or seeds are gathered, as in a year of scarcity when serious.General long-grained nonglutinous rice wound
Evil overweights round-grained rice glutinous rice, and late rice injury overweights early rice.Its host further includes dryland rice, wild rice, water chestnut is white and Leersia Sw is in addition to rice
Natural host plant, but it is not very common to fall ill on these plants.In addition there is water chestnut white and the sheath chaff of Leersia is careless, batch shell
Grass, moleplant seed and maggot grass etc..
At present, bacterial blight of rice from 1884 Japan since, in succession Asia, Europe, Oceania, Africa and
North America is found.Its occurrence scope oneself extend over the entire globe rice cropping area, in countries in the world morbidity on the books country just
There are Japan, the U.S., China waits 27 countries wherein with Japan, and India and China's morbidity are more serious.
Xanthomonas oryzae pv. oryzicola belongs to Prokaryota (Procaryotes), gracilicute door (Gracilicutes), Huang Dan
The rice Xanthomonas campestris of born of the same parents Cordycepps xanthomonas (Xanthomonas).Germ size is about 1.0-2.5 μm or 0.4-0.6 μm.It is single
Born of the same parents are paired once in a while, but do not form gemma and pod membrane into chain, have the raw single flagellum in end, can move about, no spore, no pod membrane.Leather
Blue Albert'stain Albert reaction negative, growth optimum temperature are 25 1 28 DEG C, and lethal temperature is 51 DEG C.It is sick on gravy peptone agar medium
Opportunistic pathogen growth is fast, and bacterium colony is in often faint yellow, and circular, smooth, regular edges are dome-shaped, thick, aerobic.The germ and rice
Though the pathogenic and phene of leaf spot bacteria is very different, its inhereditary feature and Physiological-biochemical Characters have very big phase again
Like property, therefore the bacterium should be used as a mutation in bacterial blight of rice strain.
Bacterial stripe, also known as slice disease, cecospora spot.It is by Xanthomonas campestris PV.oryzicola (Xanthomonas oryzae
Pv.oryzicola a kind of important quarantine seed-borne disease of rice caused by).Main cause harm rice leaf and young age blade.
It can all occur from seedling stage to heading stage, but be more with tillering stage to heading morbidity early period.And the germ can not only be attached to seed
Surface can also be deposited with Interior Seed sometimes.Scab is confined to parenchyma cell between vein, just translucent small for bottle green water soaking mode
Point becomes faint yellow slat spot between vein to upper and lower extension quickly, not wide due to being limited scab by vein, under a few cases
It is extending transversely that germ can cross vein.Scab is very short on disease-resistant variety, size about 1x10mm, and scab is longer in susceptible variety, has
Shi Keda 30-50mm. or longer, many scabs can be linked to be the withered spot of bulk.Scab both ends are in infiltrative type green.Chang Yi on scab
Go out a large amount of beading yellow bacterial ooze, jelly granule after doing.Bacterium overflows and seldom seldom sees on bacterial leaf spot scab, and bacterial stripe
On be then often covered with small pearl bacterium liquid.In addition to rice, there are Australia rice, broad-leaved rice, water chestnut white and many wild rices etc. can be by
It infects and falls ill, such as Oryza latifolia, Oryza brachyantha, Oryza breviligulate etc..Exist in recent years
Also there is large area on the late hybrid rice of the Yangtze river basin.Bacterial leaf streak of rice is maximum to the harmfulness of long-grained nonglutinous rice, have gone up for
Main bacteria disease on south China, Central-South rice region.
At present, bacterial leaf streak of rice is mainly distributed on subtropical and tropical zones, such as Philippine, Thailand, India,
From the point of view of Area distribution, Asia and Africa are concentrated mainly on.In China, bacterial leaf streak of rice is mainly distributed on southern provinces and regions,
Such as Guangdong, Guizhou, Yunnan, Sichuan, Zhejiang, Jiangxi, Hunan, Hubei.
2007, the Ministry of Agriculture of China promulgated《The People's Republic of China (PRC) enters the territory plant quarantine harmful organism register》, it is public
Cloth includes harmful insect, mollusk, fungi, prokaryotes, nematode and virus etc., totally 435 kinds of harmful organisms.Wherein infect
The seed-borne disease of rice crop be mainly xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola),
Rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) etc..Both germs can not only infect rice crop
And it can be transferred through infected seed and carry out long-distance communications and by means of wind and rain, irrigation water and entomochory and farming operations
Closely causes agricultural output to decline for the progress such as cutter, vessel, greatly hinders China's agricultural development
At present, Symptom Observation is to carry out pathogenic to detect a kind of common method, and Symptom Observation method is by directly seeing
The symptom feature at hazard of plant position is examined to achieve the purpose that detection.Pathology types mainly have:It wilts, spot ulcer, soft rot,
Deformity and scab etc..The position of morbidity, the scope of symptom variation and different onset individual are important to note that during Symptom Observation
Between symptom characteristic comparison, but some disease pathogeneticing characteristics are but extremely similar, only with being visually difficult to differentiate between, when two kinds or more
It is also difficult to distinguish that such as xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria are only difficult with naked eyes during the same plant of disease infestation
To distinguish, only it is more difficult to distinguish with Symptom Observation in Combined Infection.
Microscopic observation is to cut fritter diseased tissues in low power Microscopic observation, and thalline such as can be observed and spray bacterium shows
As then can determine whether as bacteriosis.It is in addition, different and affine with all kinds of dyestuffs using the microscopic structure of different bacterium
Power difference can also accurately identify the strain infected.But for the similar different germs of microscopic structure, microscope
Observation is just difficult to differentiate between the species of germ, such as xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria.
Utilize PCR (polymerase chain reaction, Polymerase Chain Reaction abbreviation PCR) method detection crop disease
Original has many advantages, such as sensitive, quick, high specificity, and specificity and sensitivity are then better than Symptom Observation and the side of being separately cultured
Method.
" probe and primer sequence applied to rice leaf spot bacteria detection that Chinese Patent Application No. is 03105358.0
Row " (its Publication No. CN1524964, publication date are on 09 01st, 2004) receive son using probe technique according to siderophore
Gene order otherness devises specific primer and probe.Using the probe to carrying disease germs rice paddy seed and blade is examined
It surveys.The probe can quickly directly identify leaf spot bacteria, and germ and core need not be separated from rice paddy seed
Acid extraction, is directly detected using seed leachate as template.It, can be with Direct Identification to Asia using the probe in detecting high specificity
Kind.Chinese Patent Application No. is 201110035412.5 " for detecting primer, method and the reagent of rice leaf spot bacteria
Box " (its Publication No. CN102618628A, publication date are on 09 01st, 2004) devises one kind for detecting the white leaf of rice
The primer of blight bacteria strain GD414, and optimize reaction system, can it is easy, promptly by GD414 and other leaf spot bacterias
Bacterial strain distinguishes.But above-mentioned patent can only single detection rice leaf spot bacteria, can not accomplish paddy bacterial streak
Germ and the Multiple detection of rice leaf spot bacteria.
Chinese Patent Application No. is 201010218670.2 " rice leaf spot bacteria, xanthomonas oryzae pv. oryzicola, mandarin orange
(its Publication No. CN101921843A, publication date are in December, 2010 to tangerine ulcer bacteria and the detection method of cabbage black rot bacterium "
22 days) by two pairs of primers simultaneously to detect sample DNA profiling carry out PCR reaction amplification obtain PCR product, then PCR produce
The specific probe hybridization set on object and genetic chip, whether according to the results of hybridization positive, to judge whether detect sample
Contain rice leaf spot bacteria, xanthomonas oryzae pv. oryzicola, citrus ulcer bacteria or cabbage black rot bacterium；The patent can be with one
Secondary property completes detection to 4 kinds of bacteriums, and testing result is simple and clear, easily determines.But the detection side of above-mentioned patent structure
Method in the DNA profiling to detecting sample also need on PCR product and genetic chip after PCR reaction amplifications obtain PCR product
The specific probe of setting is hybridized, according to results of hybridization come determine detection as a result, the time-consuming effort of detection process and into
This is higher.It can not accomplish quickly to detect.
" rice leaf spot bacteria and the X. c. pv. oryzicola bacterium that Chinese Patent Application No. is 200910030127.7
Padlock probes and multiple detection method " (its Publication No. CN101608235, publication date are on December 23rd, 2009) is directed to
Rice leaf spot bacteria and X. c. pv. oryzicola bacterium respectively devise a padlock probe, and combination Macroaary technologies can be same
When detect rice leaf spot bacteria and Xanthomonas campestris PV.oryzicola, have stronger specificity, sensitivity and stability, be the white leaf of rice
The detection of blight bacterium and Xanthomonas campestris PV.oryzicola provides quick, sensitive, special technical method.The detection of above-mentioned patent structure
The padlock probes head and the tail that its testing principle of method is first to make the state of " unlocking " with desmoenzyme are combined with DNA profiling, then with company
The padlock probes that enzyme elimination is not completely combined with DNA profiling are connect, and make to be combined right-on " unlocking " shape with DNA profiling
The connection of state padlock probes head and the tail circlewise, finally expands the cricoid padlock probes of enzymatic amplification with large fragment.Complete reaction
Process.Detected through gel electrophoresis finally may be employed or macroarray carries out hybridization check.Detection process is cumbersome to be taken, and
Testing cost is higher.Can not accomplish detection it is quick, operation it is simple.
The content of the invention
Xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola), rice leaf spot bacteria
(Xanthomonas oryzae pv.oryzae) is all the seed-borne disease for mainly infecting rice crop, often infects same plant simultaneously
Object.Present invention aim to address it is existing for simultaneously detect both cause of diseases technical operation it is cumbersome, take time and effort, cost compared with
The technical issues of high, it is a kind of accurate for both cause of diseases energy to provide, quickly, simple, lower-cost multiplex PCR detection reagent
Box, multi-PCR detection method and its primer special.
Technical scheme is as follows：
1. a kind of primer special X-b of quick detection rice leaf spot bacteria, it is characterised in that：The detection rice is white
The primer special X-b of leaf spoting bacteria is made of sense primer and anti-sense primer, and detection the special of rice leaf spot bacteria is drawn
The base sequence of the sense primer of object X-b such as SEQ ID NO：Shown in 1, the primer special X- of the detection rice leaf spot bacteria
The base sequence of the anti-sense primer of b such as SEQ ID NO：Shown in 2, target product fragment length is 461bp.
2. a kind of primer special X-t of quick detection xanthomonas oryzae pv. oryzicola, it is characterised in that：The detection water
The primer special X-t of rice Xanthomonas campestris PV.oryzicola is made of sense primer and anti-sense primer, the detection paddy bacterial streak
The base sequence of the sense primer of the primer special X-t of germ such as SEQ ID NO：Shown in 3, the detection paddy bacterial streak
The base sequence of the anti-sense primer of the primer special X-t of germ such as SEQ ID NO：Shown in 4, target product fragment length is
348bp。
3. it is a kind of for the kit of quick detection xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria simultaneously, it is special
Sign is that the kit includes following optimal multi-PRC reaction system and optimum reaction condition：
The optimal multi-PRC reaction system is：The MgCl of 10 × PCR reaction buffers 2.5 μ l, 25mM21.5 μ l,
The 2 μ l of dNTP Mixture of 2.5mM；Each 0.5 μ of upstream and downstream primer of the primer special X-b of 1 μM of detection rice leaf spot bacteria
L, the Taq of each 0.5 μ l, 5U/ μ l of upstream and downstream primer of the primer special X-t of 1 μM of detection xanthomonas oryzae pv. oryzicola
Polymerase 0.5 μ l, 1 × 105Cfu/mL~1 × 1080.5 μ l, the RNase Free of rice paddy seed sample to be checked of cfu/mL
dH2O complements to 25 μ l；
The base sequence such as SEQ ID NO of the sense primer of the primer special X-b of the detection rice leaf spot bacteria：1
It is shown, the base sequence such as SEQ ID NO of the anti-sense primer of the primer special X-b of the detection rice leaf spot bacteria：2 institutes
Show, target product fragment length is 461bp；
The base sequence such as SEQ ID of the sense primer of the primer special X-t of the detection xanthomonas oryzae pv. oryzicola
NO：Shown in 3, the base sequence such as SEQ ID of the anti-sense primer of the primer special X-t of the detection xanthomonas oryzae pv. oryzicola
NO：Shown in 4, target product fragment length is 348bp；
The optimum reaction condition is：
95 DEG C of pre-degeneration 5min, from 95 DEG C of denaturation 30sec, 56 DEG C of annealing 60sec, 72 DEG C extend 60sec operations 30 and follow
Ring, 72 DEG C of extension 10min.
4. the molecular biology method of a kind of while quick detection xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria,
It is characterized by comprising following steps：
(1) preparation of rice paddy seed sample to be checked
Rice paddy seed to be checked takes 20 phosphate buffers for adding in 5ml sterilizings, is crushed in the mortar of sterilizing and extraction is made
Liquid, places 5h whirlpool mixing 5min at 25 DEG C, and the phosphate buffer for adding sterilizing is diluted to 100 times of suspension, takes 100
28 DEG C of culture 50h on tablet microlitre are placed in, using the sterile water process corresponding time to compare；By the lawn on tablet with sterile washing
It takes off, obtains 1 × 105cfu/mL-1×108The rice paddy seed sample to be checked of cfu/mL；
(2) multi-PRC reaction
Multi-PRC reaction system is：The MgCl of 10 × PCR reaction buffers 2.5 μ l, 25mM2The dNTP of 1.5 μ l, 2.5mM
Mixture 2μl；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, 1 μM of detection water
0.5 μ of Taq polymerase of each 0.5 μ l, 5U/ μ l of upstream and downstream primer of the primer special X-t of rice Xanthomonas campestris PV.oryzicola
L, 1 × 105cfu/mL-1×1080.5 μ l, the RNase Free dH of rice paddy seed sample to be checked of cfu/mL2O complements to 25 μ l；
The base sequence such as SEQ ID NO of the sense primer of the primer special X-b of the detection rice leaf spot bacteria：1
It is shown, the base sequence such as SEQ ID NO of the anti-sense primer of the primer special X-b of the detection rice leaf spot bacteria：2 institutes
Show, target product fragment length is 461bp；
The base sequence such as SEQ ID of the sense primer of the primer special X-t of the detection xanthomonas oryzae pv. oryzicola
NO：Shown in 3, the base sequence such as SEQ ID of the anti-sense primer of the primer special X-t of the detection xanthomonas oryzae pv. oryzicola
NO：Shown in 4, target product fragment length is 348bp；
Multi-PRC reaction condition is：
95 DEG C of pre-degeneration 5min, from 95 DEG C of denaturation 30sec, 56 DEG C of annealing 60sec, 72 DEG C extend 60sec operations 30 and follow
Ring, 72 DEG C of extension 10min；
(3) electrophoresis detection and judgement
After multi-PRC reaction terminates, 5 μ l PCR products are taken, amplification is detected with 1.5% agarose electrophoresis, if
It is consistent with the target product fragment length predicted to clip size, then with corresponding bacterium in the rice paddy seed sample to be checked.
Compared with prior art, the beneficial effects of the invention are as follows：
Multiple PCR detection kit, the multi-PCR detection method of the present invention, by standard bacteria rice leaf spot bacteria
Selection with the target fragment of xanthomonas oryzae pv. oryzicola, design, annealing temperature and the time of two pairs of primer specials, extension temperature
Degree and time, the parameter designings such as dosage of each reacted constituent, specificity is especially when so that the present invention is detected both germs at the same time
By force, inspection result is accurate, inspection operation simplifies, and need to only judge PCR product clip size and the target product fragment length of prediction
Whether accurate testing result can be unanimously drawn immediately, without being cloned, being sequenced again and sequence alignment, overcome existing skill
Art also needs to hybridize PCR product with the specific probe set on genetic chip, detection process not only time-consuming effort but also cost
It is higher or the padlock probes head and the tail for first making the state of " unlocking " with ligase is needed to be combined with DNA profiling in the prior art, then use
The padlock probes that excision enzyme elimination is not completely combined with DNA profiling, and make to be combined with DNA profiling right-on " unlocking "
The connection of state padlock probes head and the tail circlewise, finally expands the cricoid padlock probes of enzymatic amplification with large fragment, completes anti-
Process to be answered, detected through gel electrophoresis finally may be employed or macroarray carries out hybridization check, detection process is cumbersome to be taken,
And the defects of testing cost is higher.
The above-mentioned prior art at least needs 4-8 hour, and two padlock probes of synthesis at least need 1600 yuan and examine
It needs to use expensive genetic chip during surveying either to be of little use and more expensive excision enzyme or large fragment amplification enzyme.Using this
Inventive method only needs 2 hours to can obtain testing result, and four primer synthesis only need 80 yuan and common PCR amplification is only needed to try
Detection can be completed in agent, without the expensive experiment consumptive material such as expensive padlock probes, reduces testing cost.Therefore, it is of the invention
New technical solution is proposed, not only specificity is special when method, kit, special primer pair two kinds of germs detect simultaneously
By force, and it is quick, accurate, judge easy, operation sequence is simple, without be cloned, be sequenced again and sequence alignment can be direct
Judge, testing cost is relatively low.
The present invention is respectively provided with important for the academic research of both germs and to the bacteria-treating of easy infection plant simultaneously
Meaning.
SEQ ID NO in sequence table：Shown in 1 is the upper of the primer special X-b of present invention detection rice leaf spot bacteria
Swim the base sequence of primer.
SEQ ID NO in sequence table：Shown in 2 is under the primer special X-b that the present invention detects rice leaf spot bacteria
Swim the base sequence of primer.
SEQ ID NO in sequence table：That shown in 3 is the primer special X-t of present invention detection xanthomonas oryzae pv. oryzicola
Sense primer base sequence.
SEQ ID NO in sequence table：That shown in 4 is the primer special X-t of present invention detection xanthomonas oryzae pv. oryzicola
Anti-sense primer base sequence.
Description of the drawings
Fig. 1 is to carry out PCR to standard bacteria rice leaf spot bacteria using the primer special X-b of detection rice leaf spot bacteria
Amplification, to obtained PCR product through 1.5% agarose gel electrophoresis result figure.M is Marker, and swimming lane 1 is negative control, is swum
Road 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 the primer special X-t using detection xanthomonas oryzae pv. oryzicola to standard bacteria bacterial leaf streak of rice
Bacterium carries out PCR amplification, to obtained PCR product through 1.5% agarose gel electrophoresis result figure.M is Marker, and swimming lane 1 is the moon
Property control, swimming lane 2 is standard bacteria rice leaf spot bacteria, and swimming lane 3 is standard bacteria xanthomonas oryzae pv. oryzicola, and target stripe is long
It spends for 348bp.
Fig. 3 is to utilize the kit of the present invention and the primer special X-b of rice leaf spot bacteria and detection rice bacterium
The primer special X-t of property Population of Xanthomonas Oryzae Pv to standard bacteria rice leaf spot bacteria, standard bacteria xanthomonas oryzae pv. oryzicola and this
The mixing sample of two kinds of standard bacterias carries out the 1.5% agarose gel electrophoresis result figure of product of multiplexed PCR amplification.In figure：M is
Marker, A and B represent each segment position horizontal in figure.Swimming lane 1 is standard bacteria xanthomonas oryzae pv. oryzicola, and swimming lane 2 is
Standard bacteria rice leaf spot bacteria, 3 standard bacteria rice leaf spot bacteria of swimming lane and standard bacteria xanthomonas oryzae pv. oryzicola mix
Bacteria liquid sample is closed, swimming lane 4 is negative control；The band of B location is standard bacteria paddy bacterial in B location and swimming lane 3 in swimming lane 1
The target stripe of Population of Xanthomonas Oryzae Pv, target stripe length are 348bp.The band of location A is standard in location A and swimming lane 3 in swimming lane 2
The target stripe of bacterium rice leaf spot bacteria, target stripe length are 461bp.
Fig. 4 is to utilize the kit of the present invention, detection method or the primer special X-b and the inspection that detect rice leaf spot bacteria
The commercially available rice paddy seed sample that the primer special X-t for surveying xanthomonas oryzae pv. oryzicola is 1 to 20 to number carries out multiplex PCR expansion
Increase gained 1.5% agarose gel electrophoresis result figure of PCR product.In figure：M is Marker, two of symbol+occur from top to bottom
Band is followed successively by the positive control of standard bacteria rice leaf spot bacteria and standard bacteria xanthomonas oryzae pv. oryzicola, and symbol-for feminine gender
Control.Swimming lane 1 to 20 is the commercially available rice paddy seed sample that number is 1 to 20, and the rice paddy seed sample of swimming lane 1 to swimming lane 20 is inspection
It surveys negative.
Fig. 5 is to utilize the kit of the present invention, detection method or the primer special X-b and the inspection that detect rice leaf spot bacteria
The commercially available rice paddy seed sample that the primer special X-t for surveying xanthomonas oryzae pv. oryzicola is 21 to 39 to number carries out multiplex PCR
Amplification gained 1.5% agarose gel electrophoresis result figure of PCR product.In figure：M is Marker, the two of symbol+occur from top to bottom
Band is followed successively by the positive control of standard bacteria rice leaf spot bacteria and standard bacteria xanthomonas oryzae pv. oryzicola, and symbol-for the moon
Property control.Swimming lane 21 to 39 is the commercially available rice paddy seed sample that number is 21 to 39, and swimming lane 22 is xanthomonas oryzae pv. oryzicola
Target stripe, target stripe length is 348bp, and the rice paddy seed sample of swimming lane 21 is negative for detection, swimming lane 23 to swimming lane 39
Rice paddy seed sample it is negative for detection.
Fig. 6 is to utilize the kit of the present invention, detection method or the primer special X-b and the inspection that detect rice leaf spot bacteria
The commercially available rice paddy seed sample that the primer special X-t for surveying xanthomonas oryzae pv. oryzicola is 40 to 47 to number carries out multiplex PCR
Amplification gained 1.5% agarose gel electrophoresis result figure of PCR product.In figure：M is Marker, the two of symbol+occur from top to bottom
Band is followed successively by the positive control of standard bacteria rice leaf spot bacteria and standard bacteria xanthomonas oryzae pv. oryzicola, and symbol-for the moon
Property control.Swimming lane 40 to 47 is the commercially available rice paddy seed sample that number is 40 to 47, and it 45 is rice leaf spot bacteria that swimming lane, which is,
Target stripe, target stripe length are 461bp.For swimming lane 40 to swimming lane 44, the rice paddy seed sample of swimming lane 46 to swimming lane 47 is inspection
It surveys negative.
Specific embodiment
The invention will be further described with reference to embodiments, facilitates a better understanding of the present invention, but is not intended to limit this
Invention.
Experimental method in the following example, is conventional method, agents useful for same, material, can from biochemical reagents shop with
And Seed Market is bought.Rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) used and rice bacterium
Property Population of Xanthomonas Oryzae Pv (Xanthomonas oryzae pv.oryzicola) is the standard bacteria being commercially available from Institute of Micro-biology.
Rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) and rice bacterium in following embodiment
Property Population of Xanthomonas Oryzae Pv (Xanthomonas oryzae pv.oryzicola) two kinds of bacteriums trained respectively by sterile beef extract-peptone
28 DEG C of constant temperature of nutrient solution (being formulated as beef extract 0.5g, peptone 1g, sodium chloride 0.5g, distilled water 100mL, pH 7.0~7.2) shake
Bed 180r/min culture 48h, are respectively prepared concentration as 1 × 108The bacterium solution and concentration of the rice leaf spot bacteria of cfu/mL for 1 ×
108The xanthomonas oryzae pv. oryzicola of cfu/mL.
The primer special X-b and its design of the quick detection rice leaf spot bacteria of the present invention of embodiment 1 and this is used to special
With the conventional molecular biological method of primer X-b detection rice leaf spot bacterias.
(1) design and synthesis of the primer special X-b of rice leaf spot bacteria is detected
The primer special X-b for detecting rice leaf spot bacteria is according to hrp in rice leaf spot bacteria DNA sequence
1 and of gene cluster, 2 region genetic fragments, carry out homology analysis with DNAMAN6.0, determine high conservative region
Domain, then the specific primer designed with 6.0 primer-design softwares of Primer, it is primer special X-b to be numbered, and detects rice
The primer special X-b of leaf spot bacteria is made of sense primer and anti-sense primer, described to detect the special of rice leaf spot bacteria
The base sequence of the sense primer of primer X-b such as SEQ ID NO：Shown in 1, the primer special of the detection rice leaf spot bacteria
The base sequence of the anti-sense primer of X-b such as SEQ ID NO：Shown in 2, target product fragment length is 461bp.
The primer special X-b is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
(2) the molecular biology side of rice leaf spot bacteria is detected with the primer special X-b of detection rice leaf spot bacteria
Method comprises the following steps：
1. synthesize the primer special X-b of the detection rice leaf spot bacteria described in above-mentioned steps (1).The white leaf of detection rice
The primer special X-b of blight bacterium is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
2. the substance PCR reactions of rice leaf spot bacteria
Using the bacterium solution of rice leaf spot bacteria as template, the bacterium solution of xanthomonas oryzae pv. oryzicola is positive control, is sterilized
Blank culture solution afterwards makees negative control, and common PCR reaction system and PCR reaction conditions are as described in table 1：95 DEG C of pre-degenerations
5min carries out 35 cycles from 94 DEG C of denaturation 30s, 58 DEG C of 30s that anneal, 72 DEG C of extension 30s, and 72 DEG C extend 10min and carry out PCR eventually
Amplification.
1 common PCR reaction system of table
In table 1：The sense primer of primer special X-b draws for the upstream of the primer special X-b of detection rice leaf spot bacteria
Object is write a Chinese character in simplified form；The anti-sense primer of primer special X-b is the letter of the anti-sense primer of the primer special X-b of detection rice leaf spot bacteria
It writes.
5. electrophoresis detection
After PCR reaction terminatings, 5 μ l PCR products are taken, 1.5% agarose electrophoresis detection amplification can amplify
The segment of 461bp or so is rice leaf spot bacteria, as shown in Figure 1.
6. clone and sequencing
It is produced with QIAquick PCR purification kits (being purchased from Qiagen Inc., Chatsworth, CA) purifying PCR amplification
Object, and connected with pGEM-T carriers (being purchased from Promega, Madison, WI, USA), pass through Escherichia coli cells
(JM-109) convert, with standard biological operation sequence, surveyed automatically with AB13370DNA by hundred Tyke biology Co., Ltd of Beijing
Sequence instrument is sequenced.
7. sequence alignment
The sequence of acquisition is passed through into NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) it compares and divides
Analysis, 1 and of rice leaf spot bacteria hrp gene cluster with (the accession number AB045311.1) of Genbank reports
The uniformity of 2 region genes is 99%.Prove the primer special X-b of the detection rice leaf spot bacteria for the white leaf of rice
Blight bacterium has good specificity.
The primer special X-t and its design of the quick detection xanthomonas oryzae pv. oryzicola of the present invention of embodiment 2 and use this
To the conventional molecular biological method of primer special X-t detection xanthomonas oryzae pv. oryzicolas.
(1) design and synthesis of the primer special X-t of xanthomonas oryzae pv. oryzicola is detected
The primer special X-t for detecting xanthomonas oryzae pv. oryzicola is according to xanthomonas oryzae pv. oryzicola chromogene
AvrRxo1-ORF1 genetic fragments in group carry out homology analysis with DNAMAN6.0, determine highly conserved region, then use
The specific primer of 6.0 primer-design softwares of Primer design, it is primer special X-t to be numbered, the detection rice bacterium
The primer special X-t of property Population of Xanthomonas Oryzae Pv is made of sense primer and anti-sense primer, the detection xanthomonas oryzae pv. oryzicola
Primer special X-t sense primer base sequence such as SEQ ID NO：Shown in 3, the detection xanthomonas oryzae pv. oryzicola
Primer special X-t anti-sense primer base sequence such as SEQ ID NO：Shown in 4, the detection xanthomonas oryzae pv. oryzicola
Primer special X-t target product fragment length be 348bp.The primer special X-t is had by Shanghai JaRa bioengineering
Limit company synthesizes.
(1) molecule of xanthomonas oryzae pv. oryzicola is detected with the primer special X-t of detection xanthomonas oryzae pv. oryzicola
Biological method comprises the following steps：
1. synthesize the primer special X-t of the detection xanthomonas oryzae pv. oryzicola described in above-mentioned steps (1).The detection rice
The primer special X-t of Xanthomonas campestris PV.oryzicola is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
2. the substance PCR reactions of xanthomonas oryzae pv. oryzicola
Using the bacterium solution of xanthomonas oryzae pv. oryzicola as template, the bacterium solution of rice leaf spot bacteria is positive control, is sterilized
Blank culture solution afterwards makees negative control, and common PCR reaction system and PCR reaction conditions are as described in table 2：95 DEG C of pre-degenerations
5min runs 35 cycles from 94 DEG C of denaturation 30s, 58 DEG C of 30s that anneal, 72 DEG C of extension 30s, and 72 DEG C extend 10min and carry out PCR eventually
Amplification.
2 common PCR reaction system of table
In table 2：The sense primer of primer special X-t is upper for the primer special X-t's of detection xanthomonas oryzae pv. oryzicola
Trip primer is write a Chinese character in simplified form；The anti-sense primer of primer special X-t is the downstream of the primer special X-t of detection xanthomonas oryzae pv. oryzicola
Primer is write a Chinese character in simplified form.
5. electrophoresis detection
After PCR reaction terminatings, 5 μ l PCR products, 1.5% agarose electrophoresis detection amplification are taken.It can amplify
The segment of 348bp or so is xanthomonas oryzae pv. oryzicola, as shown in Figure 2.
6. clone and sequencing
It is produced with QIAquick PCR purification kits (being purchased from Qiagen Inc., Chatsworth, CA) purifying PCR amplification
Object connects with pGEM-T carriers (being purchased from Promega, Madison, WI, USA), and passes through Escherichia coli cells
(JM-109) convert, with standard biological operation sequence, surveyed automatically with AB13370DNA by hundred Tyke biology Co., Ltd of Beijing
Sequence instrument is sequenced.
7. sequence alignment
The sequence of acquisition is passed through into NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) it compares and divides
Analysis, one with (accession number AY395713.1) xanthomonas oryzae pv. oryzicola AvrRxo1-ORF1 genes of Genbank reports
Cause property is 99%.Prove the primer special X-t of the detection xanthomonas oryzae pv. oryzicola for rice bacterial leaf streak of rice
Bacterium has good specificity.
Embodiment 3 provided by the present invention for quick and precisely detecting xanthomonas oryzae pv. oryzicola simultaneously
(Xanthomonas oryzae pv.oryzicola) and rice leaf spot bacteria (Xanthomonas oryzae
Pv.oryzae kit) includes：
10 × PCR reaction buffers,
The MgCl of 25mM2,
The dNTP Mixture of 2.5mM,
The sense primer of the primer special X-b of 1 μM of detection rice leaf spot bacteria,
The anti-sense primer of the primer special X-b of 1 μM of detection rice leaf spot bacteria,
The sense primer of the primer special X-t of 1 μM of detection xanthomonas oryzae pv. oryzicola,
The anti-sense 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 dH2O；
Equipped with specification in the kit, the optimal multi-PRC reaction body that the useful present invention described in specification recommends
System and optimum reaction condition, the optimal multi-PRC reaction system is as shown in table 3, and optimum reaction condition is：95 DEG C of pre-degenerations
5min runs 30 Xun Huans from 95 DEG C of denaturation 30sec, 56 DEG C of annealing 60sec, 72 DEG C of extension 60sec；72 DEG C of extension 10min.
The optimal multi-PRC reaction system of 3 invention of table
In table 3：
The sense primer of primer special X-b is the letter of the sense primer of the primer special X-b of detection rice leaf spot bacteria
It writes；
The anti-sense primer of primer special X-b is the letter of the anti-sense primer of the primer special X-b of detection rice leaf spot bacteria
It writes；
The sense primer of primer special X-t is the sense primer of the primer special X-t of detection xanthomonas oryzae pv. oryzicola
Write a Chinese character in simplified form；
The anti-sense primer of primer special X-t is the anti-sense primer of the primer special X-t of detection xanthomonas oryzae pv. oryzicola
Write a Chinese character in simplified form；
The base sequence such as SEQ ID NO of the sense primer of the primer special X-b of the detection rice leaf spot bacteria：1
It is shown, the base sequence such as SEQ ID NO of the anti-sense primer of the primer special X-b of the detection rice leaf spot bacteria：2 institutes
Show, target product fragment length is 461bp；
The base sequence such as SEQ ID of the sense primer of the primer special X-t of the detection xanthomonas oryzae pv. oryzicola
NO：Shown in 3, the base sequence such as SEQ ID of the anti-sense primer of the primer special X-t of the detection xanthomonas oryzae pv. oryzicola
NO：Shown in 4, target product fragment length is 348bp；
Embodiment 4 is with the multiple PCR detection kit and detection method of the present invention respectively to rice leaf spot bacteria, water
The detection of the mixing sample of rice Xanthomonas campestris PV.oryzicola, rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola
1. respectively with rice leaf spot bacteria, xanthomonas oryzae pv. oryzicola, rice leaf spot bacteria and paddy bacterial
The bacterium solution of Population of Xanthomonas Oryzae Pv biased sample is template, and it is negative right to make culture solution work with the blank beef extract-peptone culture solution after sterilizing
According to.Respectively with table 3 described in optimal multi-PRC reaction system (rice paddy seed sample to be checked is respectively bacterial blight of rice at this time
The bacteria liquid sample of bacterium, the bacteria liquid sample of xanthomonas oryzae pv. oryzicola, rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola
Bacterium solution mixed in equal amounts sample) and reaction condition be：95 DEG C of pre-degeneration 5min；From 94 DEG C of denaturation 30sec, 56 DEG C of annealing
60sec, 72 DEG C of extension 60sec run 30 Xun Huans；72 DEG C of extension 10min carry out multiplexed PCR amplification.
2. after PCR reaction terminatings, taking 5 μ l PCR products, amplification is detected with 1.5% agarose electrophoresis.It can distinguish
The segment for amplifying 461bp or so, 348bp or so and 461bp or so and 348bp or so combinations is to be believed that successfully to detect
Rice leaf spot bacteria, xanthomonas oryzae pv. oryzicola, the aggregate sample of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola
This is as shown in Figure 3.What swimming lane 1 can will be apparent that from Fig. 3 sees the band that length is 348bp, and what swimming lane 2 can will be apparent that sees
See the band that length is 461bp, length that swimming lane 3 can will be apparent that see is the band of 348bp and 461bp, and the moon of swimming lane 4
Property control without band, explanation：With the multiple PCR detection kit and multi-PCR detection method of the present invention respectively to standard bacteria water
Rice bacterial leaf spot pathogenic bacteria, xanthomonas oryzae pv. oryzicola, the mixing sample of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola
The accuracy of detection.
Embodiment 5 detects 47 kinds of commercially available rice with the multiple PCR detection kit and multi-PCR detection method of the present invention
The Carriage of seed (each rice paddy seed is by the following method)
(1) preparation of rice paddy seed sample to be checked
Each rice paddy seed to be checked takes 20 phosphate buffers for adding in 5ml sterilizings, is thoroughly pressed in the mortar of sterilizing
It is broken that extracting solution is made, 5h whirlpool mixing 5min are placed at 25 DEG C, the phosphate buffer for adding sterilizing is diluted to 100 times outstanding
Supernatant liquid takes 100 microlitres to be placed in 28 DEG C of culture 50h on tablet, using the sterile water process corresponding time as control.By the lawn on tablet
It is eluted with sterile water, obtains 1 × 105cfu/mL-1×108The rice paddy seed sample to be checked of cfu/mL；The phosphoric acid buffer
Liquid and preparation method thereof：NaCl 80g, Na2HPO4·2H2O 32.3g, NaH2PO4·2H2O 4.5g add distilled water 885mL, use HCl/
NaOH tune pH to 7.2, last constant volume are 1000mL.
(2) multi-PRC reaction
The 0.5 μ l of rice paddy seed sample to be checked described in step (1) are taken in PCR reaction tubes, are separately added into 10 × PCR reactions
The MgCl of buffer solution 2.5 μ l, 25mM2The 2 μ l of dNTP Mixture of 1.5 μ l, 2.5mM；1 μM is detected rice leaf spot bacteria
Each 0.5 μ l of upstream and downstream primer of primer special X-b, the primer special X-t's of 1 μM of detection xanthomonas oryzae pv. oryzicola is upper and lower
Swim 0.5 μ l, the RNase Free dH of Taqpolymerase of each 0.5 μ l, 5U/ μ l of primer2O complements to 25 μ l；The detection water
The base sequence of the sense primer of the primer special X-b of rice bacterial leaf spot pathogenic bacteria such as SEQ ID NO：Shown in 1, the detection rice is white
The base sequence of the anti-sense primer of the primer special X-b of leaf spoting bacteria such as SEQ ID NO：Shown in 2, target product fragment length is
461bp；The base sequence such as SEQ ID NO of the sense primer of the primer special X-t of the detection xanthomonas oryzae pv. oryzicola：
Shown in 3, the base sequence such as SEQ ID NO of the anti-sense primer of the primer special X-t of the detection xanthomonas oryzae pv. oryzicola：
Shown in 4, target product fragment length is 348bp.
Reaction condition is：95 DEG C of pre-degeneration 5min, from 94 DEG C of denaturation 30sec, 56 DEG C of annealing 60sec, 72 DEG C of extension 60sec
30 Xun Huans of operation；72 DEG C of extension 10min.
(3) electrophoresis detection and judgement
After multi-PRC reaction terminates, 5 μ l PCR products are taken, amplification is detected with 1.5% agarose electrophoresis, if
It is consistent with the target product fragment length predicted to clip size, then with corresponding bacterium in the seed sample to be checked；Not on the same day
In triplicate.
Testing result such as Fig. 4, Fig. 5, Fig. 6：Result is not identical in triplicate on the same day.
The experimental results showed that：In the swimming lane for representing 47 kinds of rice paddy seed samples, swimming lane 22 detected paddy bacterial
The target stripe 348bp of Population of Xanthomonas Oryzae Pv, swimming lane 45 detected the target stripe 461bp of rice leaf spot bacteria, other swimming lanes
It is negative for detection.Result is not identical in triplicate on the same day.This explanation is in 47 kinds of rice paddy seeds of detection, corresponding No. 22 seed belts
There is xanthomonas oryzae pv. oryzicola, No. 45 seeds carry rice leaf spot bacteria.Show：Multiple PCR detection kit of the present invention
And multi-PCR detection method of the present invention need not be cloned, is sequenced and sequence alignment again, obtain clip size and prediction target
Segment is consistent, it is possible to determine to detect rice leaf spot bacteria (Xanthomonas simultaneously with corresponding bacterium in the sample
Oryzae pv.oryzae) and two kinds of diseases of xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola)
The specific especially strong, testing result of bacterium accurately, quickly, only 2 hours need to can obtain testing result, detect program simplicity,
Reduce testing cost.
<110>Yunnan Prov Agriculture University
Yunnan Lin Peng agricultural science and technologys Co., Ltd
<120>The multiple PCR detection kit of two kinds of seed-borne diseases and its detection of primer special and multiplex PCR on rice
Method
<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 (1)
1. the molecular biology method of a kind of while quick detection xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria, special
Sign is to comprise the following steps：
(1) preparation of rice paddy seed sample to be checked
Rice paddy seed to be checked takes 20 phosphate buffers for adding in 5ml sterilizings, is crushed in the mortar of sterilizing and extracting solution is made,
5h whirlpool mixing 5min are placed at 25 DEG C, the phosphate buffer for adding sterilizing is diluted to 100 times of suspension, takes 100 microlitres
28 DEG C of culture 50h on tablet are placed in, using the sterile water process corresponding time as control；By the lawn on tablet under sterile water elution
Come, obtain 1 × 105cfu/mL-1×108The rice paddy seed sample to be checked of cfu/mL；
(2) multi-PRC reaction
Multi-PRC reaction system is：The MgCl of 10 × PCR reaction buffers 2.5 μ l, 25mM2The dNTP of 1.5 μ l, 2.5mM
Mixture 2μl；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, 1 μM of detection water
0.5 μ of Taq polymerase of each 0.5 μ l, 5U/ μ l of upstream and downstream primer of the primer special X-t of rice Xanthomonas campestris PV.oryzicola
L, 1 × 105cfu/mL-1×1080.5 μ l, the RNase Free dH of rice paddy seed sample to be checked of cfu/mL2O complements to 25 μ l；
The base sequence such as SEQ ID NO of the sense primer of the primer special X-b of the detection rice leaf spot bacteria：Shown in 1,
The base sequence such as SEQ ID NO of the anti-sense primer of the primer special X-b of the detection rice leaf spot bacteria：Shown in 2, target
Product sheet segment length is 461bp；
The base sequence such as SEQ ID NO of the sense primer of the primer special X-t of the detection xanthomonas oryzae pv. oryzicola：3
It is shown, the base sequence such as SEQ ID NO of the anti-sense primer of the primer special X-t of the detection xanthomonas oryzae pv. oryzicola：4
Shown, target product fragment length is 348bp；
Multi-PRC reaction condition is：
95 DEG C of pre-degeneration 5min, from 95 DEG C of denaturation 30sec, 56 DEG C of annealing 60sec, 72 DEG C extend 60sec operations 30 and cycle, and 72
DEG C extension 10min；
(3) electrophoresis detection and judgement
After multi-PRC reaction terminates, 5 μ l PCR products are taken, amplification are detected with 1.5% agarose electrophoresis, if obtaining piece
Duan great little is consistent with the target product fragment length predicted, then with corresponding bacterium in the rice paddy seed sample to be checked.
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| CN117737275A (en) * | 2021-12-31 | 2024-03-22 | 杭州飞时达生物科技有限公司 | Primer group and kit for detecting bacterial leaf blight pathogenic bacteria of rice based on LAMP |
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