CN102914564A - Detection method for rice kernel smut - Google Patents
Detection method for rice kernel smut Download PDFInfo
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- CN102914564A CN102914564A CN2012103070558A CN201210307055A CN102914564A CN 102914564 A CN102914564 A CN 102914564A CN 2012103070558 A CN2012103070558 A CN 2012103070558A CN 201210307055 A CN201210307055 A CN 201210307055A CN 102914564 A CN102914564 A CN 102914564A
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Abstract
The invention belongs to the technical fields of biometric identification and automatic detection, and discloses a detection method for rice kernel smut. The detection method comprises the following steps: preparing a standard product and a product to be detected, wherein the standard product is a rice grain without the rice kernel smut germs and a rice grain with gradiently diluted rice grain brand spores, and the product to be detected is a rice grain to be detected; detecting the standard product and the product to be detected by adopting an electronic nose through a direct headspace inspiration process; extracting a sensor characteristic value of the electronic nose to obtain a standard sample and a sample to be detected; analyzing the standard sample and the sample to be detected by adopting a clustering method so as to judge what kind of the standard sample and the sample to be detected belongs to, thereby judging whether the sample to be detected contains the rice kernel smut germs and the rice grain brand spores or not. According to the method, the electronic nose is only applied to detection and the clustering method is applied to judgment, so that the detection time is shortened, the detection cost is reduced and the accuracy is improved.
Description
Technical field
The invention belongs to bio-identification and Automatic Measurement Technique field, be specifically related to the method for paddy rice rice kernel smut bacterium in a kind of applying electronic nose technology fast detecting paddy rice, rice, the rice husk.
Background technology
Paddy rice is one of China's staple food crop, and cultivated area accounts for 1/4 of national cultivated area, and annual production accounts for 1/2 of national total grain output.The harm of rice disease seriously affects Rice Production always.Prevent and treat under the condition existing, the whole nation reaches 2,000,000 tons because various rice diseases cause the paddy underproduction every year on average, if do not prevent and treat, the underproduction can reach 3,000,000 tons.
The rice disease kind is a lot, and the whole world has nearly hundred kinds, and there are more than 70 kinds that reach of formal record in China, and what wherein have Economic Importance has more than 20 to plant.It is large that area occurs in rice blast (Rice blast), rice sheath blight disease (Rice sheath blight), bacterial blight of rice (Rice bacterial leaf blight), rice bacterial stripe (Rice bacterial leaf streak), Kernel Smut of Rice (Rice kernel smut), popular strong, harm is serious.
Wherein, the rice kernel smut that is caused by paddy rice rice kernel smut bacterium (Tilletia barclayana) claims again black smut, is commonly called as black grain paddy, Wu Migu etc., is a kind of fungal disease.Since 20 century 70s were promoted hybrid rice mid-term, along with the raising of breeding of hybrid rice output, this is sick to occur more extensively, and its extent of injury is also increasing, just progressively rises to one of important disease on the paddy rice.No matter be in the production of hybrid seeds or the large Tanaka of Rice Production, this disease has generation.General infected seed rate 10%～20%, seriously ill field has a strong impact on output and the seed quality of the production of hybrid seeds up to more than 50%.
Paddy rice rice kernel smut bacterium mainly be by on the seed with teleutospore propagate, seed is the important source of infection of Second Year morbidity in spite of illness, strengthens the check to rice paddy seed, prevents that in spite of illness it is the essential measure of this disease spread of prevention that seed rice is called in without the lesion.Mainly be to carry out microscopic examination by sampling indoor to the check of this disease in the seed, but the method complex operation, detection time of needing when sample is more are long, degree of accuracy is not high, and damages sample.Such as standard GB/T/T 28079-2011 " paddy rice rice kernel smut bacterium quarantine identification method ", stipulated the method for paddy rice rice kernel smut bacterium Morphological Identification.But, actual for producing, be badly in need of simple to operate, detection time is short, degree of accuracy is high, do not damage measuring means and the technology of sample.
Electronic Nose (Electronic nose) is a kind of electronic device that can identify simple or complicated smell, formed by smell sampling operation device, gas sensor array and three kinds of function elements of signal processing system, can monitor continuously, in real time the smell situation of ad-hoc location.Each sensor in the array has different sensitivity to tested gas, for example, No. one gas can produce high response at certain sensor, then be low-response to other sensors, equally, the sensor of No. two high responses of γ-ray emission is then insensitive to a gas, and namely whole sensor array is different to the response data of gas with various, just this difference just makes system identify smell according to the response data of sensor.
The core devices of Electronic Nose is gas sensor, and gas sensor can be divided into a lot of types such as metal oxide type, galvanochemistry type, conductive polymer type, mass type, photoionization type according to the difference of principle, and most widely used is the metal oxide type.The application scenario of Electronic Nose comprises environmental monitoring, product detection, medical diagnosis, explosive detection etc.
After the paddy rice grain of rice was subjected to rice kernel smut harm, the sick grain of rice was all or part of destroyed, and becoming the Powdered thing of lividity is the teleutospore of pathogen.Compare with the normal grain of rice, produced other material, with special odor, this just provides possibility for the rice kernel smut bacterium that utilizes Electronic Nose Technology to detect in the paddy rice.At present, there is not yet the research report of applying electronic nose technology fast detecting paddy rice rice kernel smut bacterium method.
Cluster analysis (Cluster analysis) is a kind of Multielement statistical analysis method that sample is classified.According to the principle of " things of a kind come together, people of a mind fall into the same group; things of a kind come together, people of a mind fall into the same group ", in the situation that can for referencely namely not have priori without any pattern, with Data classification to different classes (or bunch), object in same bunch has very large similarity, and the object between different bunches has very large diversity.
Oneself successfully is applied to a plurality of fields of bio-science clustering method, as to animals and plants classification, gene order classification, protein properties classification etc., a large amount of available Software of Fuzzy Clustering Analysis occurred, is more convenient for its promotion and application.At present, there is not yet the research report of using clustering method express-analysis paddy rice rice kernel smut bacterium method.
Summary of the invention
1. goal of the invention.
In order to simplify paddy rice rice kernel smut bacterium is detected action required, reduce and detect required time, improve the degree of accuracy that detects rice kernel smut bacterium teleutospore number, and realize the invention provides the method for paddy rice rice kernel smut bacterium in a kind of applying electronic nose technology fast detecting paddy rice, rice, the rice husk to detecting paddy rice grain of rice sample nondestructive.
2. technical scheme.
A kind of paddy rice rice kernel smut bacterium detection method as shown in Figure 1, comprises the steps:
Step 1: sample preparation, comprise the preparation of standard items and product to be tested, standard items are the paddy rice grain of rice of the rice kernel smut bacterium teleutospore number that do not contain the paddy rice grain of rice of rice kernel smut bacterium and gradient dilution, product to be tested is the paddy rice grain of rice that whether contains rice kernel smut bacterium and rice kernel smut bacterium teleutospore number to be detected, take by weighing the quantitative paddy rice grain of rice, join in the container, and sealing;
Step 2: the mensuration mode, the applying electronic nose is measured standard items and product to be tested, adopts the empty gettering of immediate roof, and namely direct sample introduction syringe needle with Electronic Nose passes container closure, enters into container, and the sample introduction syringe needle is positioned at paddy rice grain of rice top;
Step 3: feature extraction, extract the sensor characteristics value of Electronic Nose, obtain master sample and sample to be tested, master sample is that the applying electronic nose is measured standard items, the sensor characteristics value of the Electronic Nose of extracting, sample to be tested is that the applying electronic nose is measured product to be tested, the sensor characteristics value of the Electronic Nose of extracting;
Step 4: analyze and judge, use cluster analysis master sample and sample to be tested, judge which in the master sample sample to be tested belong to, be product to be tested with standard items in which is identical or the most close, thereby judge whether product to be tested contains rice kernel smut bacterium and rice kernel smut bacterium teleutospore number.
Further, the paddy rice grain of rice of the rice kernel smut bacterium teleutospore number of above-mentioned steps 1 described gradient dilution refers to that per 50 gram rice kernel smut bacterium teleutospore numbers are respectively 10
5, 10
4, 10
3, 10
2, 10
1, 10
0The individual paddy rice grain of rice.
Further, above-mentioned steps 4 described application clustering methods, when calculating similarity, the index that adopts is one or more in Euclidean distance (Euclid distance), mahalanobis distance (Mahalanobis distance), the related coefficient (Correlation coefficient), and preferred version is three and combines.
3. beneficial effect.
Paddy rice rice kernel smut bacterium in applying electronic nose technology fast detecting paddy rice of the present invention, rice, the rice husk is compared with existing detection method, and the present invention has following technical advantage:
(1) simple to operate.Only need the sample introduction syringe needle of Electronic Nose is passed container closure, enter into the container that the grain of rice is housed, do not need other complex operations, such as pathogenicbacteria separation or pathogen DNA extraction etc.
(2) detection time is short.Saved the steps such as pathogenicbacteria separation or DNA extraction, single pattern detection required time only is about 2 minutes.
(3) cost is low.Do not need other reagent in the testing process, can not produce any infringement to product to be tested, greatly saved testing cost.
(4) degree of accuracy is high.Can detect per 50 gram paddy rice rice kernel smut bacterium teleutospore numbers and be the sample about 10.
As seen, this method is practical, can satisfy the needs of rice paddy seed check, is adapted at large-scale promotion application in the rice paddy seed check.
Description of drawings
Fig. 1 is the flow chart of steps of method.
Fig. 2 is principal component analysis (PCA) (PCA) analytical standard sample results figure, and wherein, CK represents in the 50 gram paddy rice grain of rice and do not contain the rice kernel smut bacterium, and 1,2,3,4,5, the 6 rice kernel smut bacterium teleutospore numbers that represent in the 50 gram paddy rice grain of rice are respectively 10
5, 10
4, 10
3, 10
2, 10
1, 10
0Individual.
Fig. 3 is linear discriminant analysis (LDA) analytical standard sample results figure, and wherein, CK represents in the 50 gram paddy rice grain of rice and do not contain the rice kernel smut bacterium, and 1,2,3,4,5, the 6 rice kernel smut bacterium teleutospore numbers that represent in the 50 gram paddy rice grain of rice are respectively 10
5, 10
4, 10
3, 10
2, 10
1, 10
0Individual.
Embodiment
Embodiment 1:
Container uses Erlenmeyer flask, and double-deck preservative film sealing is adopted in sealing.
(1) sample preparation.Take by weighing the 50 gram paddy rice grain of rice, put in 100 milliliters the Erlenmeyer flask, utilize blood counting chamber, at microscopically the smut bacterium teleutospore concentration of the paddy rice grain of rice is made into 10
8Individual/milliliter.Gradient dilution adds and is equipped with in the triangular flask of the paddy rice grain of rice, makes the smut bacterium teleutospore number difference 10 in per 50 gram paddy rice grain of rice
5, 10
4, 10
3, 10
2, 10
1, 10
0Individual and clear water contrasts, and forms 7 standard items, and wherein 1 is the paddy rice grain of rice that does not contain the rice kernel smut bacterium, and 6 is the paddy rice grain of rice of the rice kernel smut bacterium teleutospore number of gradient dilution in addition, respectively with double-deck preservative film sealing.In addition, the paddy rice grain of rice that whether contains rice kernel smut bacterium and rice kernel smut bacterium teleutospore number to be detected joined in 100 milliliters the triangular flask, form 1 product to be tested, with double-deck preservative film sealing.
(2) mensuration mode.Electronic Nose is measured and is adopted the empty gettering of immediate roof, and namely direct sample introduction syringe needle with Electronic Nose passes double-deck preservative film, enters into Erlenmeyer flask, and the sample introduction syringe needle is positioned at paddy rice grain of rice top; Sampling time is 1 second/group, and the sensor self-cleaning time is 100 seconds, and the sensor return-to-zero-time is 10 seconds, and the preparation of samples time is 3 seconds, and the sample introduction flow is 300 ml/min, and the analytical sampling time is 40 seconds.
(3) feature extraction.7 standard items are extracted respectively 10 sensor values, as master sample; 1 product to be tested is extracted 10 sensor values, as sample to be tested.The PEN3 type Electronic Nose that adopts German AIRSENSE company to produce contains 10 different metal oxide sensors.
(4) analyze judgement.Use cluster analysis master sample and sample to be tested, when calculating similarity, the index that adopts is that Euclidean distance, mahalanobis distance and three of related coefficients combine, less according to Euclidean distance and mahalanobis distance, related coefficient large and close to or equal 1, judge which in the master sample sample to be tested belong to, be product to be tested with standard items in which is identical or the most close, thereby judge whether testing sample contains rice kernel smut bacterium and rice kernel smut bacterium teleutospore number.
Use principal component analysis (PCA) (PCA) analytical standard sample, the result as shown in Figure 2, wherein, CK represent in the 50 gram paddy rice grain of rice and does not contain the rice kernel smut bacterium, 1,2,3,4,5,6 represent the 50 rice kernel smut bacterium teleutospore numbers that restrain in the paddy rice grain of rice is respectively 10
5, 10
4, 10
3, 10
2, 10
1, 10
0Individual.Among the figure CK and 1,2,3,4,5,6 separately independent, do not have overlapping, show that the paddy rice grain of rice (CK) that does not contain the rice kernel smut bacterium and the paddy rice grain of rice (1,2,3,4,5,6) that contains rice kernel smut bacterium teleutospore are easy to make a distinction, simultaneously, rice kernel smut bacterium teleutospore number also has certain separating capacity.
Use linear discriminant analysis (LDA) analytical standard sample, the result as shown in Figure 3, wherein, CK represent in the 50 gram paddy rice grain of rice and does not contain the rice kernel smut bacterium, 1,2,3,4,5,6 represent the 50 rice kernel smut bacterium teleutospore numbers that restrain in the paddy rice grain of rice is respectively 10
5, 10
4, 10
3, 10
2, 10
1, 10
0Individual.CK and 1,2,3,4,5,6 does not have overlapping among the figure, show that the paddy rice grain of rice (CK) that does not contain the rice kernel smut bacterium and the paddy rice grain of rice that contains rice kernel smut bacterium teleutospore are easy to make a distinction, but 2, overlap between 3,4,5,6, show rice kernel smut bacterium teleutospore is counted separating capacity not as good as principal component analysis (PCA).
The result of synthesizing map 2 and Fig. 3 shows that selected standard items are that reasonably the applying electronic nose is measured standard items, obtains master sample, and it is effective whether containing the rice kernel smut bacterium for the differentiation standard items.
Above-described embodiment does not limit the present invention in any way, and every employing is equal to replaces or technical scheme that the mode of equivalent transformation obtains all drops in protection scope of the present invention.
Claims (7)
1. a paddy rice rice kernel smut bacterium detection method is characterized in that comprising the steps:
Step 1: sample preparation, comprise the preparation of standard items and product to be tested, standard items are the paddy rice grain of rice of the rice kernel smut bacterium teleutospore number that do not contain the paddy rice grain of rice of rice kernel smut bacterium and gradient dilution, product to be tested is the paddy rice grain of rice that whether contains rice kernel smut bacterium and rice kernel smut bacterium teleutospore number to be detected, take by weighing the quantitative paddy rice grain of rice, join in the container, and sealing;
Step 2: the mensuration mode, the applying electronic nose is measured standard items and product to be tested, adopts the empty gettering of immediate roof, and namely direct sample introduction syringe needle with Electronic Nose passes container closure, enters into container, and the sample introduction syringe needle is positioned at paddy rice grain of rice top;
Step 3: feature extraction, extract the sensor characteristics value of Electronic Nose, obtain master sample and sample to be tested, master sample is that the applying electronic nose is measured standard items, the sensor characteristics value of the Electronic Nose of extracting, sample to be tested is that the applying electronic nose is measured product to be tested, the sensor characteristics value of the Electronic Nose of extracting;
Step 4: analyze and judge, use cluster analysis master sample and sample to be tested, judge which kind of in the master sample sample to be tested belong to, be product to be tested with standard items in which is identical or the most close, thereby judge whether product to be tested contains rice kernel smut bacterium and rice kernel smut bacterium teleutospore number.
2. detection method according to claim 1, it is characterized in that: the paddy rice grain of rice of the rice kernel smut bacterium teleutospore number of above-mentioned steps 1 described gradient dilution refers to that rice kernel smut bacterium teleutospore number is respectively 10
5, 10
4, 10
3, 10
2, 10
1, 10
0The individual paddy rice grain of rice.
3. detection method according to claim 1, it is characterized in that: above-mentioned steps 1 described container is Erlenmeyer flask.
4. detection method according to claim 1 is characterized in that: double-deck preservative film sealing is adopted in above-mentioned steps 1 described sealing.
5. detection method according to claim 1, it is characterized in that: above-mentioned steps 2 described applying electronic noses are measured standard items and product to be tested, actual conditions is to be 1 second/group in the sampling time, the sensor self-cleaning time is 100 seconds, the sensor return-to-zero-time is 10 seconds, the preparation of samples time is 3 seconds, and the sample introduction flow is 300 ml/min, and the analytical sampling time is 40 seconds.
6. detection method according to claim 1 is characterized in that: the PEN3 type Electronic Nose that above-mentioned steps 3 described Electronic Nose adopt German AIRSENSE company to produce, contain 10 different metal oxide sensors, and obtain 10 sensor characteristics values.
7. detection method according to claim 1, it is characterized in that: above-mentioned steps 4 described application clustering methods, when calculating similarity, the index of employing is one or more in Euclidean distance, mahalanobis distance, the related coefficient, and preferred version is three and combines.
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Application publication date: 20130206 |