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Primer for detecting the soybean phytophthora and kit and method thereof

A technology of Phytophthora sojae and a kit, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve the ITS sequence difference of Phytophthora species, reduce the sensitivity of PCR reaction, Phytophthora and its close relatives Distinguish between related species and other issues

Inactive Publication Date: 2012-09-05
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, more and more studies have found that the ITS sequences of some Phytophthora species have very little difference, and it is difficult to distinguish these Phytophthora from their relatives with primers designed for this target
When Wang et al. (2006) designed Phytophthora sojae-specific primers based on the ITS sequence, they found that the similarity between Phytophthora sojae and the ITS sequence of P.melonis was as high as 97%, and the 3' end of the specific primer of Phytophthora sojae was only similar to that of P.melonis The corresponding sequence of Phytophthora differs by 1 base, and the annealing temperature needs to be raised above 66°C to distinguish the two Phytophthora, but too high annealing temperature will reduce the sensitivity of PCR reaction
And increasing the annealing temperature can not completely solve this problem, when the concentration of P.melonis is higher, it will still cause false positive phenomenon

Method used

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  • Primer for detecting the soybean phytophthora and kit and method thereof
  • Primer for detecting the soybean phytophthora and kit and method thereof
  • Primer for detecting the soybean phytophthora and kit and method thereof

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Effect test

Embodiment 1

[0032] 1. Primer design and synthesis

[0033] According to the Phytophthora sojae transposon sequence (see figure 1 ) design a pair of specific primers TrapF1 / TrapR1 (see the nucleotide sequence table SEQ ID NO.1 and SEQ ID NO.2) for the molecular detection of Phytophthora sojae, in order to improve the sensitivity of detection, the present invention is based on the sequence design Another pair of primers, TrapF2 / TrapR2 (see the nucleotide sequence table for details, SEQ ID NO.3 and SEQ ID NO.4), was used as the second round of amplification primers for nested PCR. All primers were synthesized by Shanghai Shenggong Company.

[0034] 2. Establish a conventional PCR reaction system

[0035] The total volume of the PCR reaction mixture is 25 μl, including: template DNA of the corresponding concentration, 0.5 μM TrapF1 / TrapR1 primers, 50 μM each of the four dNTPs, 2.5 μl 10×PCR reaction buffer, 2 mM Mg 2+ , 2.5 μl of 1% BSA, 1.25 units of Taq enzyme (TaKaRa), and the amplifica...

Embodiment 2

[0039] Extract DNA from various samples as templates for PCR reactions. The specific process is as follows:

[0040] 1. Extraction of mycelium powder DNA

[0041] Refer to the method of Sambrook et al. (1989) and slightly improve it. Take a small amount of mycelium powder, add 900 μl 2% CTAB extract and 90 μl 10% SDS, vortex and mix well, put in a water bath at 55°C for 1 hour, and invert several times every 10 minutes. Centrifuge at 12000rpm for 10min, take the supernatant and add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), mix by inverting, centrifuge at 12000rpm for 10min; transfer the supernatant to a new tube, add an equal volume of chloroform, and mix gently by inverting Evenly, centrifuge at 12000rpm for 5min. Transfer the supernatant to a new tube, add 2 times the volume of absolute ethanol and 1 / 10 volume of 3M NaAc (pH 5.2), and precipitate at -20°C (>1h). Centrifuge at 12000 rpm for 10 min, pour off the supernatant, wash the precipitate twice ...

Embodiment 3

[0052]A total of 232 strains of 26 Phytophthora species and 29 other species were amplified by PCR using eukaryotic general primers ITS1 / ITS4 and specific primers TrapF1 / TrapR1 respectively. See Table 1. All the strains in the test are monospore strains, which are preserved in the Department of Plant Pathology, Nanjing Agricultural University. The results showed that the eukaryotic universal primer ITS1 / ITS4 could amplify a band of about 700bp from all tested strains (see Table 1). It proves that the DNA proposed in this study can be used for PCR amplification, excluding the influence of DNA quality on the amplification results. Specific primer TrapF1 / TrapR1 can only specifically amplify a 267bp band from 120 P.sojae bacterial strains tested (please see Table 1, Figure 2-4 ). According to the Phytophthora sojae transposon sequence (see figure 1 ) designed primers have species specificity and can distinguish P.sojae from other Phytophthora species and fungi.

[0053] Table...

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Abstract

The invention provides a primer for detecting the soybean phytophthora, and a kit and a method thereof. The primer and the detecting method for detecting the soybean phytophthora have high accuracy, high specificity and high sensitivity, and can detect the various propagules of the soybean phytophthora such as hypha, oospore, zoospore and the like. The whole technique is fast, sensitive and accurate, which has important significance in the prevention of foreign soybean phytophthora, the detection of pathogenic bacteria in domestic affected area, the international trade and the like.

Description

technical field [0001] The invention relates to a primer, a kit and a method for detecting Phytophthora soybean, belonging to the field of biotechnology. Background technique [0002] Phytophthora root rot caused by Phytophthora sojae Kaufmann&Gerdemann infecting soybeans is one of the devastating diseases in soybean production, and it is also a class of quarantine objects announced by my country (Xu Zhigang, 2003). Phytophthora sojae can infect soybeans at various stages of soybean growth, and the disease is particularly serious in wet and rainy seasons. Because Phytophthora sojae reproduces sexually in a homologous manner, a large number of oospores can be produced on diseased plants. Plants The oospores left in the soil after harvesting become the primary infection source of Phytophthora sojae, and it is reported that the oospores of Phytophthora sojae can survive in the soil for more than 5 years (Xu Xiuhong et al., 2003). Therefore, once Phytophthora soybean root rot o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 王源超郑小波董莎萌孟军
Owner NANJING AGRICULTURAL UNIVERSITY
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