Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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

A kind of spot pathogen and bacterial technology, which is applied in the field of plant protection and molecular biology detection, can solve the problems of time-consuming, difficult cross-reaction of antibodies, inability to adapt to rapid detection, etc.

Inactive Publication Date: 2017-11-10
安徽出入境检验检疫局检验检疫技术中心 +2
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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
Among the serum detection methods, ELISA is the most widely used method, which can detect a large number of samples in a short time, but it is not easy to obtain antibodies from different strains, and cross-reactions during the reaction process can easily cause false positive or false negative phenomena.
PCR detection technology can quickly and effectively detect rice bacterial stripe disease from rice seed samples, which is of great significance to domestic plant quarantine and to ensure the export of rice seeds and rice in my country. Detection technology is applied to the detection and identification of streak bacteria, but a reliable diagnosis cannot be made for some samples with low bacterial carrier rate, resulting in false negative results

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method and kit for detecting xanthomonas oryzae pv. oryzicola by use of digital-PCR (polymerase chain reaction)
  • Method and kit for detecting xanthomonas oryzae pv. oryzicola by use of digital-PCR (polymerase chain reaction)
  • Method and kit for detecting xanthomonas oryzae pv. oryzicola by use of digital-PCR (polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Design of primer pairs and specific probe combinations for the digital PCR detection of rice bacterial streak bacterium

[0031] Three primer pairs and specific probe combinations for digital PCR detection of rice bacterial streak were designed according to the putative membrane protein gene of rice bacterial streak:

[0032] Upstream primer Xoc163F: 5′-GTGTTGACCCGCGCCCTGTTCTG-3′

[0033] Downstream primer Xoc163R: 5′-CGACGACGACGGCAAGTGAGTGAC-3′

[0034]Specific probe Xoc163P: 5'-FAM-TTTCAAAGGGCAGGGCTATCTGCT-TAMRA-3'

[0035] The 5' end of the probe Xoc163P has a FAM fluorescent label, and the 3' end has a TAMRA fluorescent label.

[0036] Upstream primer Xoc163F: 5′-GTGTTGACCCGCGCCCTGTTCTG-3′

[0037] Downstream primer Xoc163R: 5′-CGACGACGACGGCAAGTGAGTGAC-3′

[0038] Specific probe Xoc163P2: 5'-FAM-CTATCTGCTGATTCCGGCAAATGT-TAMRA-3'

[0039] The 5' end of the probe Xoc163P2 has a FAM fluorescent label, and the 3' end has a TAMRA fluorescent label.

[004...

Embodiment 2

[0045] Example 2 Establishment of a Digital PCR Detection Method for Bacterial Spot of Rice

[0046] 1. Preparation of reaction system Reaction system 20ul, consisting of 2×SuperMix 10ul, primer Xoc163F 1ul, primer Xoc163R 1ul, probe Xoc163P 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.

[0047] 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.

[0048] 3. After completing step 2, transfer the droplet system to a 96-well plate and seal the film;

[0049] 4. After completing step 3, place the 96-well plate in a PCR machine for PCR amplification reaction. The reaction program: pre-denatu...

Embodiment 3

[0052] Example 3 Carry out specific detection according to the method established in Example 2

[0053] Genomic DNA of seven bacterial strains obtained from the NCPPB collection bank was extracted with a bacterial genomic DNA extraction kit.

[0054] According to the method of Example 2, specific detection was carried out. See the experimental results figure 1 (A04 is X.oryzaepv.oryzicola, B04 is X.oryzae pv.oryzae, C04 is X.axonopodis pv.citri, D04 is X.axonopodis pv.vesicatoria, E04 is B.glumae, F04 is A.avenae subsp.avenae , G04 is P. agglomerans, H04 is blank control). The results show that only X.oryzae pv.oryzicola produces positive micro-droplets, and other bacterial strains and blank controls do not produce positive micro-droplets (at this time, the threshold for judging is 5928, and the micro-droplet instrument below the threshold is judged as negative, and above the threshold The microdrop instrument judged as positive). Therefore, only the X.oryzae pv.oryzicola ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a method and kit for detecting xanthomonas oryzae pv. oryzicola 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 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 detection primers are designed on basis of assumed membrane protein genes of xanthomonas oryzae pv. oryzicola. Experiments prove that the provided method for detecting the xanthomonas oryzae pv. oryzicola 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 stripe disease. Background technique [0002] Rice bacterial streak (Xanthomonas oryzae pv. oryzicola), is a bacterium of the Xanthomonas family and the genus Xanthomonas, which can infect multiple species of Oryza sp. and Zizania aquatica ). In 1990, Swings et al. named this species as X.oryzae pv.oryzicola X. oryzae pv. oryzicola. The disease first occurred in the Philippines in 1918, and is currently mainly distributed in tropical and subtropical regions, such as the Philippines, Thailand, India, Pakistan, Indonesia, Bangladesh, Senegal, Nigeria, Cambodia, Vietnam, Malaysia, Mauritania, China, Australia , Nepal, Colombia, Madagascar, Sri Lanka and Cameroon; in terms of regional distribution, they are mainly concentrated in Asia and Africa. my country is mainly distributed in southern ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2563/159
Inventor 李云飞田茜王明生赵文军
Owner 安徽出入境检验检疫局检验检疫技术中心
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com