Molecular detection method aiming at Asia fusarium and application thereof

Abstract

The invention relates to a molecular detection method aiming at Asia fusarium and an application thereof. The method comprises the following steps of: carrying out PCR (polymerase chain reaction) amplification on sample-extracted DNA (deoxyribonucleic acid) by use of a pair of specific primers to Asia fusarium, and carrying out electrophoresis gel imaging analysis on an amplified product, wherein the pair of specific primers are an upstream primer SEQ ID NO.1 and a downstream primer SEQ ID NO.2; in the step of carrying out electrophoresis gel imaging analysis on the PCR amplified product, judging whether the sample is correlated with the Asia fusarium or not according to the condition of whether a specific stripe exists at 172bP or not; and amplifying a polymorphic SRAP (sequence-related amplified polymorphism) label by virtue of the pair of specific primers based on the related sequence of the Asia fusarium, wherein the sequence of the SRAP label is SEQ ID NO.3, and the size of the SRAP label is 172bp. The method provided by the invention can be used for identifying the Asia fusarium, or monitoring the pollution of the Asia fusarium to crops or feeds.

Description

technical field

[0001] The invention relates to a molecular detection method for Fusarium asiaticus and its application, and belongs to the technical field of crop disease diagnosis and pathogenic bacteria identification. Background technique

[0002] Wheat scab (Fusarium head blight, FHB) caused by Fusarium spp. is a widespread worldwide disease, which mainly occurs in warm and humid regions, and is one of the important factors affecting high, stable and high-quality wheat yields. one. Since the 1890s, barley and wheat scab have been severely affected in North America, Europe, Asia and other places. The winter wheat area in the middle and lower reaches of the Yangtze River in southern my country is serious. With global warming, changes in farming systems and farming methods, wheat scab is spreading to the Huanghuai wheat area, northern wheat area, and southwest and northwest wheat area (Ma et al., 2008), and spread to almost all wheat growing areas. In 2003, 60-80% of the...

Examples

Embodiment 1

[0026] According to the identified 42 Fusarium sub-Asian and 30 Fusarium non-Asian species, the amplification and comparison of Fusarium sub-Asian and non-Asian Fusarium were carried out by using sequence-related amplified polymorphism (SRAP) markers. pair analysis. The amplified SRAP markers are based on SRAP polymorphism markers of introns and exons in the gene sequence, and such polymorphism markers often represent polymorphisms (that is, differences) between genes. From nearly 400 markers, we found SRAP markers that can only be amplified in Fusarium subascensus but not in non-Asian Fusarium species. The sequence of the SRAP marker is SEQ ID NO.3, and its size is 172bp. By sequencing this marker and comparing it in NCBI's GeneBank, it was found that this is a new gene that only exists in Fusarium sub-Asia, and no homologous sequence has been found in the sequences of other Fusarium. We redesigned the primers for this marker.

[0027] Design a primer pair specific to the m...

Embodiment 2

[0053] The collected Fusarium strains were typed using specific primers.

[0054] Fusarium strains were collected from 7 provinces and autonomous regions in China, and the inventor defined them as: 0901, 0914, 0919, 0920, 0922, 0923, 0924, 0925, 0950, 0968, 0978, 0938, 0943, 0956 , 0966, 0822, K11, 2E22, 2041, 0825, and the collection places corresponding to each strain are recorded in Table 2.

[0055] The strain DNA sample was extracted using the improved SDS method, adding 200 μL of extraction buffer (4% SDS, 100 mM Tris-HCl, 10 mM EDTA) to an appropriate amount of mycelia, warming at 65 ° C for 30 min, and then quickly moving it into an ice bath for 30 min, adding 200 μ L of chloroform-iso Pentanol (24:1) extract was shaken and mixed, centrifuged at 12,000 rpm for 5 min, and 120 μL of isopropanol was added to the supernatant to precipitate DNA, ddH 2 O dissolves DNA. Take 10-20ng DNA into the PCR reaction system.

[0056] The PCR amplification reaction, amplification pr...

Embodiment 3

[0063] Surveillance of wheat contamination by Fusarium asiaticus.

[0064] Wheat kernels were collected from diseased wheat kernels and healthy wheat kernels from Taizhou and Wujin wheat districts in Jiangsu Province, Lu'an wheat districts in Anhui Province, Shangcheng and Huangchuan wheat districts in Henan Province, and Xiangfan wheat districts in Hubei Province.

[0065] DNA Extraction from Wheat Kernels

[0066] Wheat grains were collected from Taizhou and Wujin Mai Districts in Jiangsu Province, Lu'an Mai District in Anhui Province, Shangcheng and Huangchuan Mai Districts in Henan Province, and Xiangfan Mai District in Hubei Province. Healthy wheat grains or diseased wheat grains were taken, freeze-dried with liquid nitrogen, ground and pulverized, and DNA was extracted by the modified CTAB method (Zhang, 2004) (same as Example 1). Take 40-50ng DNA into the PCR reaction system.

[0067] The DNA of the wheat sample was subjected to the PCR amplification reaction, the amp...

technical field

[0001] The invention relates to a molecular detection method for Fusarium asiaticus and its application, and belongs to the technical field of crop disease diagnosis and pathogenic bacteria identification. Background technique

[0002] Wheat scab (Fusarium head blight, FHB) caused by Fusarium spp. is a widespread worldwide disease, which mainly occurs in warm and humid regions, and is one of the important factors affecting high, stable and high-quality wheat yields. one. Since the 1890s, barley and wheat scab have been severely affected in North America, Europe, Asia and other places. The winter wheat area in the middle and lower reaches of the Yangtze River in southern my country is serious. With global warming, changes in farming systems and farming methods, wheat scab is spreading to the Huanghuai wheat area, northern wheat area, and southwest and northwest wheat area (Ma et al., 2008), and spread to almost all wheat growing areas. In 2003, 60-80% of the...

Examples

Embodiment 1

[0026] According to the identified 42 Fusarium sub-Asian and 30 Fusarium non-Asian species, the amplification and comparison of Fusarium sub-Asian and non-Asian Fusarium were carried out by using sequence-related amplified polymorphism (SRAP) markers. pair analysis. The amplified SRAP markers are based on SRAP polymorphism markers of introns and exons in the gene sequence, and such polymorphism markers often represent polymorphisms (that is, differences) between genes. From nearly 400 markers, we found SRAP markers that can only be amplified in Fusarium subascensus but not in non-Asian Fusarium species. The sequence of the SRAP marker is SEQ ID NO.3, and its size is 172bp. By sequencing this marker and comparing it in NCBI's GeneBank, it was found that this is a new gene that only exists in Fusarium sub-Asia, and no homologous sequence has been found in the sequences of other Fusarium. We redesigned the primers for this marker.

[0027] Design a primer pair specific to the m...

Embodiment 2

[0053] The collected Fusarium strains were typed using specific primers.

[0054] Fusarium strains were collected from 7 provinces and autonomous regions in China, and the inventor defined them as: 0901, 0914, 0919, 0920, 0922, 0923, 0924, 0925, 0950, 0968, 0978, 0938, 0943, 0956 , 0966, 0822, K11, 2E22, 2041, 0825, and the collection places corresponding to each strain are recorded in Table 2.

[0055] The strain DNA sample was extracted using the improved SDS method, adding 200 μL of extraction buffer (4% SDS, 100 mM Tris-HCl, 10 mM EDTA) to an appropriate amount of mycelia, warming at 65 ° C for 30 min, and then quickly moving it into an ice bath for 30 min, adding 200 μ L of chloroform-iso Pentanol (24:1) extract was shaken and mixed, centrifuged at 12,000 rpm for 5 min, and 120 μL of isopropanol was added to the supernatant to precipitate DNA, ddH 2 O dissolves DNA. Take 10-20ng DNA into the PCR reaction system.

[0056] The PCR amplification reaction, amplification pr...

Embodiment 3

[0063] Surveillance of wheat contamination by Fusarium asiaticus.

[0064] Wheat kernels were collected from diseased wheat kernels and healthy wheat kernels from Taizhou and Wujin wheat districts in Jiangsu Province, Lu'an wheat districts in Anhui Province, Shangcheng and Huangchuan wheat districts in Henan Province, and Xiangfan wheat districts in Hubei Province.

[0065] DNA Extraction from Wheat Kernels

[0066] Wheat grains were collected from Taizhou and Wujin Mai Districts in Jiangsu Province, Lu'an Mai District in Anhui Province, Shangcheng and Huangchuan Mai Districts in Henan Province, and Xiangfan Mai District in Hubei Province. Healthy wheat grains or diseased wheat grains were taken, freeze-dried with liquid nitrogen, ground and pulverized, and DNA was extracted by the modified CTAB method (Zhang, 2004) (same as Example 1). Take 40-50ng DNA into the PCR reaction system.

[0067] The DNA of the wheat sample was subjected to the PCR amplification reaction, the amp...

Images