KR20140048548A - Species-specific oligonucleotides for detecting fusarium proliferatum by pcr and the kit comprising the same - Google Patents

Abstract

The present invention is characterized in that oligonucleotides for detecting species-specific F. proliferatum by PCR include SEQ ID NO: 1 (TPRO1) and SEQ ID NO: 2 (TPRO2). In addition, the present invention is characterized in that the kit for detecting F. proliferatum comprising the oligonucleotide set.
The primer combination according to the present invention can quickly detect and identify F. proliferatum strains contaminated with various grain samples.

Description

Species-specific oligonucleotides for detecting Fusarium proliferatum by PCR and the kit comprising the same}

The present invention relates to oligonucleotides for PCR detection of F. proliferatum , more specifically F. proliferatum occurs in corn A novel species specific oligonucleotide (primer) combination and kit comprising the same that can distinguish a strain from strains belonging to other fumonicin production presumed G. fujikuroi complexes.

G. fujikuroi species complex is a group of filamentous fungal fungal fungi with an incomplete generation of Fusarium , a phytopathogenic bacterium that causes widespread contamination of rice, corn, sorghum, millet and other crops, causing major diseases (Leslie et al. , 1990; Leslie and summerell, 2006). Species belonging to this group were divided into mating populations (MPs), depending on whether they were crossed between strains (Leslie and Summerell, 2006; Leslie et al., 2007). However, for species that exist only in the asexual generation, it was not possible to distinguish them according to their mating communities. In order to overcome this, we conducted a phylogenetic study through comparative analysis of DNA sequences, and it was confirmed that at least 40 phylogenetic species exist in the population so far (O'Donnell et. al., 1998). In addition, all species in the previous mating population were consistent with grouping according to phylogenetic species, and the efficacy of species identification using phylogeny was tested. Some of these species produce mycotoxins from the polyketide family called fumonisins in diseases and contaminants, as well as plant diseases, causing serious poisoning in humans and livestock (Marasas et. al., 1998; Nelson et al., 1993 Munkvold and Desjardins, 1997; Marasas, 2001). Fumonisin, in particular, has gained more attention as a carcinogen candidate as the pathologic link between contaminated grain intake and human esophageal cancer has emerged (Yoshizawa et al., 1994; Marasas, 2001).

F. proliferatum is a species belonging to the G. fujikuroi species complex hybrid group D (MP D, full generation: G. intermedia ), which is frequently detected in corn, wheat and barley together with F. verticillioides belonging to the G. fujikuroi complex in corn, wheat and barley, and sometimes black points. Causes symptoms. In addition, this plant produces various toxins such as fumonicin, monoliformin, beavericin, and fusaric acid, and has a relatively high concentration of fumoni in the optimal environment for toxin production. It is known to produce gods. However, unlike F. verticillioides , F. proliferatum is detected in a variety of plants, including bananas, pines, asparagus, palm trees, and eggs. Thus, F. proliferatum, except the host range is very similar to the F. verticillioides etc. mycological characteristics, mycotoxin producing ability, but the situation is very low compared to basic research F. verticillioides. In addition, the two species are very similar in morphology, making it almost impossible to distinguish them by microscopic observation and culture characteristics.

The present invention was derived to solve the above problems, the production of F. proliferatum strains produced in the corn and rice of Korea different from the production of fumonisin G. fujikuroi It is an object to provide a novel specific primer combination that can be distinguished from strains belonging to the species complex.

It is another object of the present invention to provide a kit for the detection of the bacterium comprising the primer combination.

In order to achieve the above object, the oligonucleotide for detecting a species-specific F. proliferatum by PCR of the present invention is characterized by including SEQ ID NO: 1 (TPRO1) and SEQ ID NO: 2 (TPRO2).

SEQ ID NO: 1 (TPRO1)

5'-CATCTTGGAAGACAACATGCT-3 '

SEQ ID NO: 2 (TPRO2)

5'-CAAGTGAAATCGGTGGGCAGA-3 '

In addition, the present invention is characterized in that the kit for detecting F. proliferatum comprising the oligonucleotide set.

Using the oligonucleotide for the species-specific F. proliferatum detection by the PCR of the present invention and a kit comprising the same, the strain can be easily distinguished from other fungi production strains from strains belonging to G. fujikuroi species complex. It can be detected easily.

1 is an arrangement of some nucleic acid sequences of the TEF1 gene isolated from Fusarium representative species used in the invention. Sequence positions of the primers TPRO1 and TPRO2 are indicated by black arrows, and various nucleic acid sequences within the primers between the species are indicated by black boxes. (// indicates discontinuity of nucleic acid sequences; F. proliferatum : isolated from the os17 strain. K41087 from strains: nucleotide sequence TEF1; F. verticilliodes: os9 isolated from strain TEF1 nucleotide sequence; F. fujikuroi: the TEF1 nucleotide sequence isolated from strain os5;:; F. subglutinans F. oxysporum TEF1 a nucleotide sequence isolated from strain os12 Isolated TEF1 sequence)
Figure 2 is a PCR amplified picture from the Fusarium species used in the present invention using primers TPRO1 and TPRO2; [Lane M: a 100bp ladder, lanes 1-5: F. verticillides , lanes 17-24: F. fujikuroi , lanes 26-27: F. subglutinans , lanes 6-16 & 28-29: F. proliferatum ].
3 shows Fusarium of various concentrations using TPRO1 and TPRO2. TEF1 targeting proliferatum genomic DNA This is an amplified picture; [M: 100bp ladder; Lane 1: mold genome 0.425 μg / μl; 10-fold serial dilution of genomic DNA of lanes 2-6].

The present invention relates to oligonucleotides for PCR detection of F. proliferatum , a novel species specific oligonucleotide (primer) combination that can be effectively distinguished from strains belonging to other fumonisin production presumed G. fujikuroi species complexes and It relates to a kit for the detection of the bacteria used.

Hereinafter, the present invention will be described in detail with reference to examples. These embodiments are illustrative and those skilled in the art will be able to make various modifications and alterations to the disclosed embodiments without departing from the technical spirit of the present invention. The scope of the present invention is not limited by these variations and modifications.

[ Example  One]

Strain collection: the G. fujikuroi species complexes as isolates from Korea, corn and rice during the 2009-2010 pre-sale received from the National Institute of the National Academy of Agricultural Sciences harmful organisms team eukaryotic translation elongation factor 1-alpha (EF1A used in this study ) Strains identified by phylogenetic analysis using gene sequencing (Kim et al., 2011) and strains received from the Center for Agricultural Genetic Resources (KACC) (Table 1).

   Table 1 PCR amplification using Fusarium species and specific primers used in the present invention

   Table 1 PCR amplification using Fusarium species and specific primers used in the present invention (cont.)

[ Example  2]

Amplification and Sequence Arrangement of EF1A Gene: Agar blocks of each strain incubated in potato agar medium (PDA) for 3 days for genomic DNA extraction of collected strains were inoculated in potato agar liquid medium (PDB). Shake incubation at 25 ° C for days. After extracting genomic DNA from the cells according to the method reported by Chi et al. (2009), F. proliferatum , F. verticilliodes , F. fujikuroi , EF1A from genomic DNA of F. subglutinas The gene was amplified. All PCR amplification primers used in this study were prepared by Bioneer (Korea), and 50 ng of template DNA and EmeraldAmpGT PCR Master Mix (2X Premix) (Takara Biomedicals, Japan) were added to 50 μl of total solution for PCR reaction. It was. PCR amplification using primer combinations derived from EF1A (EF1 and EF2) (Table 2) was performed under the following conditions: 30 cycles of 94 ° C 30 seconds, 55 ° C 30 seconds, 72 ° C 1 minute after 94 ° C 2 minutes of denaturation. Reaction, extended reaction of 72 ° C. for 10 minutes. After confirming PCR amplification of a single size (~ 600bp) through electrophoresis, the amplified DNA was purified using a PCR purification kit (NucloGen, Korea).

In order to determine the base sequence of the amplified DNA, a direct sequencing method was mainly used, using a primer amplification primer combination as the base sequence primer. However, if the results of the sequences are not clear, cloning the amplified DNA into the pGEM-T Easy Vector System (Promega, USA) according to previously reported methods (Hong et al., 2010), then combining the T7 and SP6 primers The base sequence was determined by. In order to confirm the final nucleotide sequence of the amplified gene, the nucleotide sequence determined by the bidirectional primers was compared by DNASTAR Corporation (USA) seqman program, and the amplified gene sequences were then sorted using the Clustral W method in the DNA STAR MEGALIGN program. .

The sequences obtained in this study can be distributed at any time by the request of the relevant researcher.

[ Example  3]

Preparation of Species-Specific Primers: To prepare F. proliferatum species-specific primers, the amplified TEF1 gene sequence was used. 680bp or less A total of 68bp of mutated sequences were identified through the alignment of the EF1A gene sequences, and TPRO1 and TPRO2 could be prepared as specific primer candidate combinations from these sites (FIG. 1). These primers were all located in the coding region of the TEF1 gene.

[ Example  4]

F. proliferatum PCR amplification using the specific primers: PCR amplification using the TRPO1 and TPRO2 combination produced above (conditions are as EF1A amplifying the above conditions, the annealing temperature is 60 ℃ used) Further, Fu monitor new generation of the three G Of the fujikuroi species complex collection strains, only 150 bp of DNA fragments were specifically amplified from the genomic DNA of F. proliferatum (Table 1, Figure 2).

To further characterize the specificity of these primers, three other Fusarium fungi (F. oxysporum , F. graminerum) PCR was performed using the genomic DNA of the template as a template, and no specific DNA fragments were detected (data not included). This confirmed the species specificity of the TPRO1 and TPRO2 primers.

Meanwhile, all F. proliferatum were subjected to PCR with rp32 and rp33 primers derived from FUM1 gene, a polyketide gene essential for fumonicin biosynthesis. DNA fragments of ˜680 bp were amplified from the genomic DNA of the strain (Table 1). F. proliferatum V116 strains produced 820 ppm fumonisin B1 and 163.9 ppm fumonisin B2 derivatives in actual rice medium (data not included). Similarly, all F. proliferatum investigated in this study Since FUM1- specific fragments were amplified from the strains, they are believed to have fumonisin production ability. However, the primer combination for amplification of FUM1 is not only F. proliferatum but also F. verticilliodes known as another fumonicin high producing strain. F. fujikuroi , also known as low or no production strain DNAs of the same size were also amplified from F. subglutinans strains (Table 1, Fig. 2). On the other hand, in the previously reported F. proliferatum amplification primer combinations PRO1 and PRO2 (Table 2), DNA fragments of expected size were amplified from F. verticilliodes as well as F. proliferatum . As a result, it was found that species specificity of TPRO1 and TPRO2 was superior to existing primer combinations. In addition, PCR was performed by diluting the F. proliferatum strain genomic DNA by 10-fold dilution from the concentration of 425 ng / μl in order to assay the detection limit of TPRO1 and TPRO2 primers, and amplifying specific primers from 0.425 pg / μl of fungal DNA. Could be (Fig. 3). Recently, the newly developed F. proliferatum specific primer by Jurado et al. (2010) was derived from the nucleotide sequence of the fumonisin biosynthetic FUM1 gene and amplified DNA fragments of expected size from all F. proliferatum . However, DNA fragments of the same size were also amplified from some of F. fujikuori and F. globosum strains. Therefore, unlike the F. proliferatum specific primers developed so far, TPRO1 and TPRO2 secured through the present invention are derived from the base sequence of the EF1A gene as well as novel, and can be said to have excellent species specificity. In addition, since the size of the amplified DNA is less than 200 bp it can be sufficiently applied to the quantitative real-time PCR (qRT-PCR) method later.

As such, the species-specific Fusarium of the present invention Oligonucleotides for the detection of proliferatum and kits comprising the same can be used to easily and easily detect the strains from grain samples by distinguishing the strains from other fumonicin production presumed G. fujikuroi complex belonging strains.

TABLE 2 Primer base sequences used in the present invention

<110> DDD, ddd <120> dsfafafa <130> sss <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <400> 1 catcttggaa gacaacatgc t 21 <210> 2 <211> 21 <212> DNA <400> 2 caagtgaaat cggtgggcag a 21

Claims (2)

Oligonucleotides for species-specific F. proliferatum detection by PCR, comprising SEQ ID NO: 1 and SEQ ID NO: 2. F. proliferatum comprising the oligonucleotides of claim 1 Detection kit.

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