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Method and kit for detecting xanthomonas oryzae pv. oryzae by use of digital-PCR (polymerase chain reaction)

A rice bacterial blight and kit technology, which is applied in the field of plant protection and molecular biology detection, can solve the problems of unreliable diagnosis, time-consuming, false positive and other problems of samples

Inactive Publication Date: 2017-11-10
CHINESE ACAD OF INSPECTION & QUARANTINE +2
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

The traditional detection method is complex and time-consuming, which can no longer meet the needs of rapid detection
ELISA is the most widely used serum detection method, which can detect a large number of samples in a short period of time, but it is not easy to obtain antibodies from different strains, and cross-reactions during the reaction process can easily cause false positives or false negatives.
PCR detection technology can quickly and effectively detect rice bacterial blight from rice seed samples. It is of great significance to domestic plant quarantine and to ensure the export of rice seeds and rice in my country. At present, there are many different PCR detection methods. The technology is applied to the detection and identification of bacterial blight, but a reliable diagnosis cannot be made for some samples with a low carrier rate, resulting in false negative results

Method used

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  • Method and kit for detecting xanthomonas oryzae pv. oryzae by use of digital-PCR (polymerase chain reaction)
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  • Method and kit for detecting xanthomonas oryzae pv. oryzae by use of digital-PCR (polymerase chain reaction)

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1 is used for the design of the primer pair and specific probe combination of rice bacterial blight digital PCR detection

[0031] According to the rhs family gene of Xanthomonas oryzae, the following 4 primer pairs and specific probe combinations for digital PCR detection of Xanthora oryzae were designed:

[0032] Upstream primer Xoo119F: 5′-AACGGATCGATGGACTTTACG-3′

[0033] Downstream primer Xoo119R: 5′-AATGTGCACTTCCGCTATGA-3′

[0034] Specific probe Xoo119P: 5'-FAM-TTCGTGTGAGGTTACCCTGC-TAMRA-3'

[0035] The 5' end of the probe Xoo119P has a FAM fluorescent label, and the 3' end has a TAMRA fluorescent label.

[0036] Upstream primer Xoo114F: 5′-GATCCGCTCGGTCGCAATGTG-3′

[0037] Downstream primer Xoo114R: 5′-GGCTCGCCCCAGTCCGTCGTA-3′

[0038] Specific probe Xoo114P: 5'-FAM-TGCAGGGTAACCTCACACGAATGG-TAMRA-3'

[0039] The 5' end of the probe Xoo114P has a FAM fluorescent label, and the 3' end has a TAMRA fluorescent label.

[0040] Upstream primer Xoo105F: ...

Embodiment 2

[0049] Example 2 Establishment of digital PCR detection method for bacterial blight of rice

[0050] 1. Preparation of reaction system Reaction system 20ul, consisting of 2×SuperMix 10ul, primer Xoo119F1ul, primer Xoo119R 1ul, probe Xoo119P 0.5ul, genomic DNA of the sample to be tested 2ul and ddH 2 O 5.5ul, genomic DNA in the blank control with ddH 2 O instead.

[0051] 2. After completing step 1, transfer the reaction system to the system sample hole of the digital PCR droplet generation card, and add 70ul of digital PCR reaction oil to the reaction oil sample hole, and transfer the digital PCR droplet generation card Transfer to the DropletGenerator micro-droplet generator, start the instrument, and get the micro-droplet system.

[0052] 3. After completing step 2, transfer the droplet system to a 96-well plate and seal the film;

[0053] 4. After completing step 3, place the 96-well plate in a PCR machine for PCR amplification reaction. The reaction program: pre-denatur...

Embodiment 3

[0056] Embodiment 3 carries out specific detection according to the method established in embodiment 2

[0057] Genomic DNA of seven bacterial strains obtained from the NCPPB collection bank was extracted with a bacterial genomic DNA extraction kit.

[0058] According to the method of Example 2, specific detection was carried out. See the experimental results figure 1 (A04 is X.oryzae pv.oryzae, B04 is X.oryzae pv.oryzicola, C04 is X.axonopodis pv.citri, D04 is X.axonopodispv.vesicatoria, E04 is B.glumae, F04 is A.avenae subsp.avenae , G04 is P. agglomerans, H04 is blank control). The results showed that only X.oryzae pv.oryzae produced positive micro-droplets, and other bacterial strains and blank controls did not produce positive micro-droplets (at this time, the threshold value as judgment was 1366, and micro-droplet instruments below the threshold value were judged as negative, and those above the threshold value were judged as negative. The microdrop instrument judged ...

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Abstract

The invention provides a method and kit for detecting xanthomonas oryzae pv. oryzae by use of digital-PCR (polymerase chain reaction). The provided kit contains a pair of specific primer pairs and a probe with fluorescent marks at two ends, wherein the nucleotide sequence of the specific primer pairs is shown in SEQ ID NO.1-2, and the nucleotide sequence of the probe is shown in SEQ ID NO.3. The primers are designed on basis of rhs family genes of xanthomonas oryzae pv. oryzae, and the method for detecting the xanthomonas oryzae pv. oryzae is good in specificity, high in sensitivity and accuracy of detection results and suitable for detection of germs in rice seed samples and has important application value.

Description

technical field [0001] The invention relates to the technical field of plant protection and molecular biology detection, in particular to a digital PCR detection method for rice bacterial blight. Background technique [0002] Rice bacterial blight (Xanthomonas oryzae pv.oryzae), is a bacterium of the Xanthomonas family and the genus Xanthomonas, which can infect rice, Tribulus terrestris, Bermudagrass, barnyardgrass, Lishihe, Grass, huskgrass, daughter, millet, paspalum, paragrass, wild rice, American wild rice, Zoysia and other Gramineae host plants, and also include Cyperaceae plants such as heteromorphous sedge and Cyperus cyperus. The pathogen was first discovered in Fukuoka, Japan in 1884, and it is the earliest bacterial disease found on rice. Since the 1950s, the scope of the disease has continued to expand. At present, rice bacterial blight has become an important disease in Asian and Pacific rice regions. In my country, rice bacterial blight was first discovered i...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/64
CPCC12Q1/686C12Q1/689C12Q2563/159
Inventor 田茜李云飞赵文军王明生
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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