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Polymerase chain reaction (PCR) detection kit for maize curvularia leaf spot and detection method for PCR detection kit

The invention relates to a technology for the detection of Curvus sp. leaf spot bacteria and a detection kit, which is applied in the field of fungal disease detection, and can solve the problems of long time consumption, inability to monitor and control the spread of pathogenic bacteria in time, and the like, and achieve the effects of high sensitivity and high accuracy.

Active Publication Date: 2012-07-18
贵州益百亿生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] An object of the present invention is to provide a PCR detection kit for Curvularia maize leaf spot to be used to solve the existing problems that the detection and identification methods of Curvularia maize leaf spot are time-consuming, unable to monitor and control the spread of pathogenic bacteria in time; the present invention Another purpose of is to provide the PCR detection method of Curvularia maize leaf spot

Method used

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  • Polymerase chain reaction (PCR) detection kit for maize curvularia leaf spot and detection method for PCR detection kit
  • Polymerase chain reaction (PCR) detection kit for maize curvularia leaf spot and detection method for PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, detection Curvularia maize leaf spot mycelium

[0048] 1) Detection kit

[0049] Design three specific primers of Curvularia zea Leaf Spot as follows to form two pairs of primers:

[0050] The first pair: upstream primer 5`-ACGGAGGATGCGGTATGG-3` (SEQ ID NO: 1)

[0051] Downstream primer 5`-CCTCGGAATCGTGCTTTT-3` (SEQ ID NO: 3)

[0052] Second pair: upstream primer 5`-GGGGCTCGCAGGCTAATGT-3` (SEQ ID NO: 2)

[0053] Downstream primer 5`-CCTCGGAATCGTGCTTTT-3` (SEQ ID NO: 3)

[0054] The above sequence was synthesized using a DNA synthesizer.

[0055] Prepare 2 sets of PCR reaction systems in the kit: the first set of reaction system consists of the following components: 1ml solution, 100μl 10×PCR reaction buffer, 20μl dNTP with a concentration of 10mM, 200 units of Taq enzyme, with a concentration of 10mM 10mM 40 μl each of the first pair of upstream and downstream primers, and the rest is ultrapure water; the second set of reaction system consists ...

Embodiment 2

[0062] Embodiment 2, detection Curvularia leaf spot bacteria in the leaf spot type leaf of corn

[0063] 1) Detection kit: use the same reaction system as in Example 1.

[0064] 2) Detection method:

[0065] One, extract pathogenic DNA from the maize leaf that produces leaf spot:

[0066] Rinse the diseased corn leaves with distilled water, absorb the water with absorbent paper; grind the leaves with liquid nitrogen and quickly transfer them to a 1.5mL centrifuge tube; add 600 μL of CTAB extract preheated for 30 minutes (65°C), and place in a 65°C water bath 30 min, shake once every 10 min, then add 500 μL chloroform:isoamyl alcohol (24:1) to the centrifuge tube, lightly pump for 10 min, centrifuge at 12000 rpm for 10 min at 4°C; take the supernatant, and then add the supernatant With the same volume of chloroform: isoamyl alcohol (24:1), lightly pump for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C; take the supernatant; add 2 / 3 volume of pre-cooled isopropa...

Embodiment 3

[0068] Embodiment 3, detection Curvularia leaf spot bacteria in necrotic corn leaves

[0069] 1) Detection kit: use the same reaction system as in Example 1.

[0070] 2) Detection method:

[0071] One, extract pathogenic DNA from the corn necrosis type leaf:

[0072] Rinse the necrotic corn leaves with distilled water, absorb the water with absorbent paper; grind the leaves with liquid nitrogen and quickly transfer them to a 1.5mL centrifuge tube; add 600 μL of CTAB extract preheated for 30 minutes (65°C), Bath in water for 30 min, shake once every 10 min, then add 500 μL chloroform: isoamyl alcohol (24:1) to the centrifuge tube, pump lightly for 10 min, centrifuge at 12000 rpm for 10 min at 4 °C; take the supernatant, and then add the supernatant Chloroform with the same volume of liquid: isoamyl alcohol (24:1), pump lightly for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C; take the supernatant; add 2 / 3 volume of pre-cooled 90% ethanol to the supernatant, an...

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Abstract

The invention relates to a polymerase chain reaction (PCR) detection kit for maize curvularia leaf spot and a detection method for the PCR detection kit. The PCR detection kit for the maize curvularia leaf spot comprises two PCR reaction systems; the first reaction system comprises a first primer pair consisting of two specific primers shown as SEQIDNO:1 and SEQIDNO:3; and the second reaction system comprises a second primer pair consisting of two specific primers shown as SEQIDNO:2 and SEQIDNO:3. The specific amplification primers are designed according to clg2p gene of the maize curvularia leaf spot, and the PCR detection kit for the maize curvularia leaf spot is constructed and applied to molecular detection of the maize curvularia leaf spot, can accurately and quickly perform molecular identification on maize curvularia leaf spot pathogenic bacteria, also can be used for detecting whether diseased maize seedlings having symptoms such as leaf spot and death are caused by the maize curvularia leaf spot, and has a great application prospect for identifying the maize curvularia leaf spot pathogenic bacteria and monitoring diseases in field.

Description

1. Technical field: [0001] The invention relates to a fungal disease detection technology in the field of biotechnology, in particular to a PCR detection kit and a detection method for Curvularia maize leaf spot bacteria. 2. Background technology: [0002] Curvularia lunata (Wakker) Boed, caused by Curvularia lunata (Wakker) Boed, has caused major damage in most countries of the world. In 1996, the disease caused severe maize loss in China, and 40% of the maize production areas were infected by the disease. The pathogen mainly harms maize leaves, and can also harm leaf sheaths and bracts. Lesions are water-soaked or light yellow translucent dots at first, and then expand into round, oval, fusiform or long strips of lesions. The shape and size of lesions are divided into 3 categories according to the variety resistance: ①Disease resistance Type lesions (R): small lesions, 1~2mm, round, oval or irregular, pale or light brown in the center, without brown rings or very thin ri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/645
Inventor 刘铜侯巨梅左豫虎慕庆峰马柄辰
Owner 贵州益百亿生物科技有限公司
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