Primer, kit and method for amplifying and typing paulownia arbuscular phytoplasma thyA gene

A gene amplification and phytoplasma technology, which is applied in the field of pathogen molecular detection and identification, can solve the problems of no phytoplasma thyA gene research reports, etc., and achieve the effect of increasing scientific research expenditure, simple kit and method, and low cost

Pending Publication Date: 2022-02-01
HEZE UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there is no research report on the thyA gene of phytoplasma

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
  • Primer, kit and method for amplifying and typing paulownia arbuscular phytoplasma thyA gene
  • Primer, kit and method for amplifying and typing paulownia arbuscular phytoplasma thyA gene
  • Primer, kit and method for amplifying and typing paulownia arbuscular phytoplasma thyA gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: PaWB thyA gene the entire length of the Witches phytoplasma portion and sequences downstream primer design

[0042] Download Onion yellows phytoplasma (OY-M, Genbank AP00628) in the NCBI, aster yellows phytoplasma (AYWB, Genbank CP000061), genomic sequences Maize bushy stunt Phytoplasma (M3, Genbank CP015149), were taken thyA gene and that performs multiple sequence alignments on the downstream portion of the sequence, the 97bp thyA gene upstream of the ORF and 95bp downstream of the downstream primer design.

[0043] PIthyAF the nucleotide sequence of the upstream primer (SEQ ID NO.1): 5'-TTAACTGCCATTTACACAGAAG-3 ';

[0044] PIthyAR nucleotide sequence of the downstream primer (SEQ ID NO.2): 5'-TTTTTAAAATATTGTAAATCTTGG-3 '.

Embodiment 2

[0045] Example 2: PaWB thyA gene obtained Witches two genotypes phytoplasma

[0046] 1. PCR for amplification method:

[0047] Paulownia disease arbuscular DNA sample as a template, using primers PIthyAF / PIthyAR PCR amplification.

[0048] PCR amplification system was: 2 × Taq PCR Mix 12.5μL, forward primer PIthyAF0.5μL, reverse primer PIthyAR 0.5μL, ddH 2 O 10μL, DNA template 1.5μL.

[0049] PCR amplification program: 94 ℃ 4 minutes; 94 ℃ 30 seconds, 53 ℃ 30 seconds, 1 minute, 35 cycles of 72 ℃; 72 ℃ 10 minutes; 10 deg.] C incubation.

[0050] Using primers PIthyAF / PIthyAR PCR reaction products were sequenced, and the sequences are aligned splicing analyzed using DNAMAN software.

[0051] 2, results analysis

[0052] In 88 parts of the Witches' sample collected in our country, PCR positive samples result 65 parts, 62 parts of the successful sequencing of the 62 samples distributed in 15 provinces and municipalities in 41 counties of the city. Sequence alignment results showed...

Embodiment 3

[0055] Example 3: Establishment and Development Kit rapid identification methods Witches Phytoplasmas PaWB thyA genotype

[0056] Comprehensive specific primers, and other factors to prevent dimer formation, amplification primers PathyA F / PathyA ORFR design PaWB ORF partial sequences of the primers amplified fragment length of 814bp.

[0057] The nucleotide sequence of the upstream primer PathyA F (SEQ ID NO.5): 5'-AAACGACCGCACTAATACAGGTACC-3 ';

[0058] The nucleotide sequence of the downstream primer PathyA ORFR (SEQ ID NO.6): 5'-TCATACAGCAATATCGCCTTTCAA-3 '.

[0059] PaWB thyA 1-genotype of DNA samples PathyA F / PathyA ORFR amplification product sequence as shown in SEQ IDNO.7.

[0060] PaWB thyA 2-genotype of DNA samples PathyA F / PathyA ORFR amplification product sequence as shown in SEQ IDNO.8.

[0061] Using DNAMAN software, respectively, and SEQ ID NO.7 SEQ ID NO.8 sequence analysis restriction endonuclease sites, such as the analysis results figure 1 and figure 2 Indi...

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 particularly relates to a primer, a kit and a method for amplifying and typing paulownia arbuscular phytoplasma PaWB thyA gene. The invention provides: (1) a PaWB thyA gene amplification method and a primer PIthyAF / PIthyAR, wherein the method and the primer can amplify the ORF full length of the PaWB thyA gene and sequences of upstream 97bp and downstream 95bp of the ORF full length; (2) a PaWB thyA gene typing primer PathyA F / PathyA ORFR, wherein the PaWB thyA gene can be divided into two genotypes by the primer; (3) two genotypes, namely, PaWB thyA-1 and PaWB thyA-2, of the gene PaWB thyA and a sequence of the gene PaWB thyA; and (4) a rapid typing kit and method for the PaWB thyA gene. Compared with a method for identifying genotypes through sequencing, the kit and the method provided by the invention are simpler, quicker and lower in cost. According to the invention, a foundation is laid for the subsequent research on the functions of the PaWB thyA genotype and the correlation between the PaWB thyA genotype and phytoplasma proliferation pathopoiesis.

Description

Technical field [0001] The present invention belongs to the field of pathogen molecular detection and identification, and therefore specifically, the primers, kits and methods of THYA gene amplification and classification of bubble columns. Background technique [0002] The boss is a class of plant pathogen prokaryotic organisms that can harm more than 1,000 plants worldwide. PAULOWNIA WITCHES '-BROOM, PAM, JUJUBEWITCHES'-BROOM, JWB, Mulberry Dwarf, MD), Wheat Blue Dwarf (Wheat Bluedwarf , WBD) is serious. Among them, the bubble clumps caused by the bubble columns in the 16SRI group were extremely widely distributed in my country, distributed in the vast range of north to the mountain customs, south to the Yangtze River Basin (Tian Etal., 1996), causing the production huge loss. Therefore, the research on the basic theory and prevention technology of planting alphabetic diseases is of great significance. [0003] The propaganda in the host plant is a certain concentration of the ...

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): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/686C12Q2600/166C12Q2521/301C12Q2565/125C12Q2537/165C12Q2545/113
Inventor 宋传生王俊刚康晓飞樊庆忠王亚丽王董梅
Owner HEZE UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap