Real-time fluorescence PCR detection primer probe combination of four Chinese fir anthracnose pathogenic bacteria, detection kit and application thereof

A technology for real-time fluorescence and primer detection

Pending Publication Date: 2022-01-07
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the PCR method requires many instruments and equipment, the sample processing is complicated, and the experimental links are too many, which is easy to introduce human errors, and takes a long time and pollutes the environment.
The design of primers for loop-mediated isothermal amplification (LAMP) technology is complex, and the type of dye used in this method will have a significant impact on sensitivity and stability, which is difficult to meet the needs of production applications
In addition, as a new isothermal nucleic acid amplification technology, recombinase-mediated isothermal amplification (RPA) has disadvantages such as long detection primers and high detection cost for a single sample.

Method used

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  • Real-time fluorescence PCR detection primer probe combination of four Chinese fir anthracnose pathogenic bacteria, detection kit and application thereof
  • Real-time fluorescence PCR detection primer probe combination of four Chinese fir anthracnose pathogenic bacteria, detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Design of primers and probes

[0036] According to primers AM-F (5'-TCATTCTACGTATGTGCCCGCCCGTTG-3') and AM-R (5'-CCAGAAATACACCGAACTTGCAAAGAT-3'), and use the primers to amplify the genomic DNA of G. gliospora anthracis, obtain the APMAT gene sequence such as SEQ ID NO.3 shows:

[0037] Primer pair 1#C-F (5′-AGCAGCGTCGATGAGGTAG-3′) / 1#C-R (5′-TCGCAGTCGGTAGGTGTAAC-3′) and probe 1#C-P (5′-FAM - CCGAGAGCGACGACACCATCAGCG-TAMRA-3').

[0038] The above primer pairs and probes were used for real-time fluorescent PCR detection of G. fir.

[0039] According to primers AM-F (5'-TCATTCTACGTATGTGCCCGCCCGTTG-3') and AM-R (5'-CCAGAAATACACCGAACTTGCAAAGAT-3'), and use the primers to amplify the genomic DNA of Anthrax fruit, obtain the APMAT gene sequence such as SEQ ID Shown in NO.4:

[0040] Primer pair MQ-F(5′-CCATCTATTCCAGCCCGATACC-3′) / MQ-R(5′-CAGTGGCGAAGAGCAACATG-3′) and probe MQ-P(5′-FAM - CCGCCGTGAGGACCATTGATTCGCC-TAMRA-3').

[0041] The above primer pairs and prob...

Embodiment 2

[0048] Embodiment 2: the detection of glyospora anthracnose bacteria, fruit anthracnose bacteria, Siamese anthracnose bacteria and Wenzhou anthracnose bacteria

[0049] 1. Strain DNA extraction

[0050] Cut 30 colonies of 1 mm in size from the test strains (Table 1) colonies that have been cultivated for 5 days 2 Put the mycelia block into 100mL CM liquid medium, and shake it on a 100rpm shaker at 25°C for 24 to 48 hours; filter it with two layers of filter membranes, recover the mycelia, and absorb the water through the filter membrane; place the mycelia in In the sterilized and cooled mortar, add liquid nitrogen and an appropriate amount of quartz sand, and quickly grind it to a fine powder; put an appropriate amount of powder in a 2mL centrifuge tube, add 800μLCTAB, shake and mix, 60°C water bath for 30min, shake once every 10min ;Add 800μLCIA, shake at 25°C 200rpm for 20min; centrifuge at 13000rpm for 10min, take 800μL supernatant into a new 2mL centrifuge tube, add an eq...

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Abstract

The invention discloses a real-time fluorescence PCR detection primer probe combination of four Chinese fir anthracnose pathogenic bacteria, a detection kit and application thereof, and belongs to the field of plant protection and quality inspection. The real-time fluorescence PCR detection primer probe combination of the four Chinese fir anthracnose pathogenic bacteria is designed for a highly conservative APMAT gene transcription interval region, and comprises colletotrichum gloeosporioides, colletotrichum fructicolum, colletotrichum siamensi and colletotrichum chrysogenum. By adopting the primer probe combination and the detection kit containing the primer probe combination, an anthracnose sample can be accurately, quickly and conveniently distinguished, whether diseased wood contains the four Chinese fir anthracnose pathogenic bacteria or not is judged, and the sensitivity reaches 1ng of template DNA. The Chinese fir anthracnose pathogenic bacteria primer probe combination and the detection kit containing the primer probe combination provide a quick, sensitive and specific method for detection of the Chinese fir anthracnose pathogenic bacteria.

Description

technical field [0001] The invention belongs to the field of plant protection and quality inspection, and more specifically relates to a combination of primers and probes for real-time fluorescent PCR detection of four kinds of Chinese fir anthracnose pathogens, a detection kit and applications thereof. Background technique [0002] Chinese fir [Cunninghamia lanceolata (Lamb.) Hook] is a unique timber tree species in China, which is widely distributed in the Yangtze River Basin and has high economic value. However, due to its single cultivar, fixed management mode, and soil fertility decline, it has led to serious occurrence of diseases and insect pests in some major cultivation areas of Chinese fir. Chinese fir anthracnose occurs year after year and is one of the most important biological disasters on Chinese fir. The pathogenic species of Chinese fir anthracnose are diverse. Current studies have found that the pathogenic bacteria mainly include Colletotrichum fructicola, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/686C12Q2563/107C12Q2561/113
Inventor 黄麟何姣王俊伟李建伟
Owner NANJING FORESTRY UNIV
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