TWI716094B - Kit and method for detection of fusarium oxysporum f. sp. melonis - Google Patents

Abstract

A kit for detection of Fusarium oxysporumf. sp. melonisis disclosed. The kit includes a primer pair and a probe. The primer pair includes a sense primer and an antisense primer with nucleotide sequences set forth as SEQ ID NOS: 3 and 4. The probe has a nucleotide sequence set forth as SEQ ID NO: 7 and has a florescent tag and a quencher tag at its two ends, respectively. The quencher tag is used to prevent the florescent tag from producing a florescent signal. Accordingly, the kit can be applied to detect whether a melon is infected by Fusarium oxysporumf. sp. melonis.

Description

Kit and method for detecting melon blight

The present invention relates to a kit for detecting pathogens, particularly a kit for detecting melon blight bacteria, and the present invention also relates to a method for detecting melon blight bacteria using the kit.

Fusarium wilt of melon (Fusarium wilt of melon) is a disease caused by the fungus Fusarium oxysporum f. sp. melonis (FOM), and it is the main limiting factor for the melon industry worldwide.

The pathogenic bacteria remaining on the melon seeds (melon blight fungus) cause the initial infection, and then the secondary infection source on the diseased plants in the field is spread by rain or airflow, causing large-scale damage. Therefore, the detection of melon blight fungus is extremely important.

The conventional method for detecting Melon blight bacteria is to use a primer pair with the nucleotide sequence shown in SEQ ID NOS: 1 and 2 to detect by the polymerase chain reaction method, but for the disease that has been infected but has not yet shown For melon seed samples with symptoms, the conventional method is still unable to effectively detect the melon blight bacteria contained therein, so there is still a need for improvement.

In order to solve the above-mentioned problems, the object of the present invention is to provide a kit for detecting melon blight bacteria, which can quickly and easily detect melon blight bacteria.

The second objective of the present invention is to provide a method for detecting melon blight bacteria, which uses the aforementioned kit to detect melon blight bacteria, so as to reduce the cost of detecting melon blight bacteria.

The kit for detecting Melon blight fungus of the present invention may include: a primer pair, including a forward primer and a reverse primer, the forward primer having the nucleotide sequence shown in SEQ ID NO: 3, The reverse primer has the nucleotide sequence shown in SEQ ID NO: 4; and a probe has the nucleotide sequence shown in SEQ ID NO: 7, and one end of the probe has a fluorescent label, The other end of the probe has a quenching mark, and the quenching mark is used to inhibit the fluorescent mark from generating a fluorescent signal.

Based on the same technical concept, the kit for detecting melon blight fungus of the present invention may also include: a primer pair including a forward primer and a reverse primer, and the forward primer has SEQ ID NO: 5, the reverse primer has the nucleotide sequence shown in SEQ ID NO: 6; and a probe having the nucleotide sequence shown in SEQ ID NO: 7, the probe One end of the needle has a fluorescent mark, and the other end of the probe has a quenching mark, and the quenching mark is used to inhibit the fluorescent mark from generating a fluorescent signal.

According to the above, by using a primer pair with a specific nucleotide sequence and a probe, the kit can be used to quickly and easily detect melon blight bacteria, and the detection result has high sensitivity and reproducibility, which is the present invention The effect.

In the kit for detecting melon blight of the present invention, the fluorescent marker is selected from at least one of FAM, TET, NED and VIC, and the quenching marker is selected from the group consisting of MGB, TAMRA and NFQ At least one; preferably, the 5'end of the probe has the fluorescent label FAM to reduce the cost of the probe; the 3'end of the probe has the quenching label MGB, so that the fluorescent Labeling FAM reduces the manufacturing cost of the probe, and makes the probe have a higher Tm value by the quenching labeling MGB, which can not only increase the template (ie, the sample DNA) that is paired with the probe, and the The difference in Tm value between the templates of the probe pairing can also increase the Tm value of the probe by the quenching label MGB, so that the length of the probe can be shortened and the manufacturing cost of the probe can be reduced; in addition, The quenching marker MGB is a non-luminescent fluorescent group, so it has a higher signal-to-noise ratio and can make the detection result more accurate.

In addition, the method for detecting melon wilt pathogen of the present invention may include: a reaction mixture preparation step of mixing a sample to be tested, a reaction buffer, a DNA mixture, as described above for detection A set of melon blight fungus and a DNA polymerase are prepared to prepare a reaction mixture, the sample to be tested contains a sample of DNA; and a lysis step is performed at 94-95°C to make the reaction mixture The sample DNA is cleaved into two single-stranded DNA. The two single-stranded DNA includes a forward single-stranded DNA and a reverse single-stranded DNA; a polymerization step is performed at 56-66°C to make the forward primer and the probe Sticking the reverse single-stranded DNA with the needle and sticking the forward single-stranded DNA with the reverse primer, and making the DNA polymerase copy the sample DNA from the forward primer, the reverse primer and the probe, At this time, the fluorescent label of the probe falls off and releases a fluorescent signal; a repeating step is to repeat the lysis step and the polymerization step in order to amplify the sample DNA; and a fluorescent detection step is to detect The fluorescent signal released after the fluorescent label of the probe falls off.

According to the above, by using the aforementioned kit for detecting melon blight bacteria, the method can quickly and easily detect melon blight bacteria, and the detection results have high sensitivity and reproducibility, which is the effect of the present invention.

In order to make the above and other objects, features and advantages of the present invention more comprehensible, the preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings:

Please refer to Figures 1a to 1c. The kit for detecting melon blight fungus according to an embodiment of the present invention may include an primer pair 1. The primer pair includes a forward primer 11 and a reverse primer 12. The forward primer 11 corresponds to the reverse single-stranded DNA T1 of Melon blight fungus, and the reverse primer 12 corresponds to the forward single-stranded DNA T2 of Melon blight fungus, so that the forward primer 11 and the reverse primer 12 can be used To jointly increase the specific gene fragments of melon blight fungus.

In addition, the kit for detecting melon blight fungus may further include a probe 2, which corresponds to the reverse single-stranded DNA T1 of the specific gene fragment amplified by the primer pair, and the probe 2 One end has a fluorescent mark 2a, and the other end has a quenching mark 2b. The quenching mark 2b is used to inhibit the fluorescent mark 2a from generating a fluorescent signal; for example, the fluorescent mark 2a may be FAM (fluorescent fluorescein amidite), TET (tetrachloro-fluorescein-CE phosphoramidite), NED (Applied Biosystems proprietary “yellow” fluorescent dye) or VIC (Applied Biosystems proprietary “green” fluorescent dye), the quenching mark 2b can be MGB (minor groove binder binder) ), TAMRA (tetramethylrhodamine) or NFQ (non-fluorescent quencher), the fluorescent marker 2a and the quenching marker 2b can also be a combination of the aforementioned selections; accordingly, the fluorescent marker will fall off during the amplification reaction, Therefore, it is no longer inhibited by the quenching mark and can release a fluorescent signal (for the convenience of explanation, the fluorescent mark that releases the fluorescent signal is hereinafter referred to as "falling off fluorescent mark 2a'", as shown in Figure 1c) .

In the first embodiment, the forward primer 11 of the primer pair 1 has the nucleotide sequence shown in SEQ ID NO: 3, and the reverse primer 12 of the primer pair 1 has the nucleotide sequence shown in SEQ ID NO: 4 In the second embodiment, the forward primer 11 of the primer pair 1 has the nucleotide sequence shown in SEQ ID NO: 5, and the reverse primer 12 of the primer pair 1 has the nucleotide sequence shown in SEQ ID NO: The nucleotide sequence shown in 6. In addition, the probe 2 has the nucleotide sequence shown in SEQ ID NO: 7, and the 5'end of the probe 2 has the fluorescent label 2a, the fluorescent label 2a is FAM, and the probe 3 The'end has the quenching mark 2b, and the quenching mark 2b is MGB and NFQ.

The kit for detecting melon blight bacteria may additionally include a reaction buffer, a deoxyribonucleic acid mixture and a DNA polymerase. The reaction buffer can be KAPA SYBR ^® FAST PCR Master Mix (available from Kapa Biosystems) 2x) set, or KAPA PROBE FAST qPCR Master Mix (2X) set from Sigma-Aldrich. The user can mix the reaction buffer, the deoxyribonucleic acid mixture, the primer pair 1 and the probe 2, and then add a sample DNA T and the DNA polymerase to the aforementioned mixture to prepare a reaction The mixed solution can make the DNA polymerase in the environment formed by the reaction buffer at a specific temperature, and use the primer pair 1 to jointly amplify the specific gene fragment. At the same time, the fluorescence of the probe 2 The mark 2a will fall off, so it is no longer suppressed by the quenching mark 2b to form a fluorescent mark 2a' that can release the fluorescent signal. The user can detect the fall off fluorescent mark 2a'. The released fluorescent signal tells whether the specific gene fragment is contained in the sample DNA T.

Therefore, the kit can be used to detect whether a test sample contains melon blight bacteria. The method can be as shown in Figure 2, including: a reaction mixture preparation step S1, a lysis step S2, a polymerization step S3, A repeat step S4 and a fluorescence detection step S5.

Specifically, the reaction mixture preparation step S1 is to mix the test sample, the reaction buffer, the deoxyribonucleic acid mixture, the primer pair 1, the probe 2, and the DNA polymerase to prepare To form the reaction mixture, the sample to be tested contains the sample DNA T.

For example, the sample to be tested can be a plastid containing the DNA T of the sample, or the sample to be tested can also be hyphae of melon blight fungus; or, the sample to be tested can be After the melon seeds are crushed, according to the journal articles published by Dellaporta et al. (Dellaprrta SL, Wood J, Hicks JB. A Plant DNA Minipreparation: Ver II. Plant Molecular Biology Reporter . 1983; 1(4):19-21.) The extracted genomic DNA (genomic DNA) is not limited here.

Please refer to Figure 1a, the lysis step S2 is to lyse the sample DNA T in the reaction mixture into two single-stranded DNAs containing the forward single-stranded DNA T2 and the reverse single-stranded DNA T1; please refer to As shown in Figure 1b, the polymerization step S3 is to make the forward primer 11 and the probe 2 adhere to the reverse single-stranded DNA T1, and make the reverse primer 12 adhere to the forward single-stranded DNA T2, and Make the DNA polymerase copy the sample DNA T from the forward primer 11, the reverse primer 12 and the probe 2, and make the fluorescent label of the probe 2 fall off and form a fall off that can release the fluorescent signal The fluorescent label 2a'; and the repeating step S4 sequentially repeats the lysis step, the bonding step, and the polymerization step to amplify the sample DNA. For example, the cracking step S2 can be performed at a temperature of 94-95°C, and the polymerization step S3 can be performed at a temperature of 56-66°C.

In the fluorescence detection step S5, the fluorescence signal released by the detached fluorescent mark 2a' is detected. For example, the user can heat up from 65°C to 99°C at a rate of 1°C every 0.05 seconds to analyze the melting curve of the amplified sample DNA, due to DNA fragments of different lengths and sequences There will be different TM values (melting temperate), and a specific peak appears at a specific temperature of the melting curve. When the melting curve and a reference curve have a similar temperature peak (±1℃), the user can determine The sample DNA in the test sample contains DNA fragments having the same length and sequence as the specific gene fragment, that is, the test sample contains melon blight fungus. Alternatively, the user can also perform the fluorescence detection step S5 immediately after each repeating step S4, and record the fluorescence signal obtained in the fluorescence detection step S5 each time, when the fluorescence signal reaches a predetermined value When the value, the number of cycles at this time is called the threshold cycle (threshold cycle, _CT value), when the _CT value ≧ 22, the user can determine that the sample DNA in the sample to be tested contains the specific gene fragment, that is, the The sample to be tested contains melon blight fungus.

Based on this, in order to verify that the primer pair and the probe can be used in combination to quickly and easily detect melon blight bacteria, and the detection results are highly sensitive and reproducible, the following experiments were performed:

(A) Construction of test mass

This experiment uses primer pairs with the nucleotide sequences described in SEQ ID NOS: 1 and 2 to amplify the chromosomal DNA of Melon blight fungus, and insert the amplified product into a pGM-T vector (purchased from GMbiolab Co) to Obtain a test plastid.

(B) Sensitivity test

This test system uses the primer pairs and probes shown in Table 1 to test the plastids, the mycelium of Melon blight fungus, and the genomic DNA extracted from the seeds of the melon as the test samples.

Table 1. Sequences of primer pairs and probes used in each group of this experiment Group Forward primer Back primer Probe B0 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 7 B1 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 7 B2 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7

The results of the sensitivity test on the test plastid, the mycelium of the melon blight fungus and the genomic DNA extracted from the melon seeds are shown in Table 2, whether it is for the test plastid, mycelium or gene. DNA, the kit of the present invention for detecting melon blight bacteria does have better sensitivity.

Table 2. Sensitivity test results of each group of samples to be tested in this experiment Group Plastids for testing (number of sets) Mycelium (μg) Genomic DNA (ng) B0 10 ⁴ 10 ^-1 2×10 ^-2 B1 10 ³ 10 ^-2 2×10 ^-2 B2 10 ² 10 ^-3 2×10 ^-3

In addition, the contaminated seeds that have been contaminated by melon blight fungus and those not contaminated by melon blight fungus are mixed to form a mixture containing different proportions of contaminated seeds, and then the plastid small amount purification reagent kit (purchased from GeneMark) is used to extract it. DNA is analyzed with the primer pairs and probes shown in Table 1, and the result is: the test sensitivity of the mixture of group B0 is 0.5%, and the test sensitivity of the mixture of B1 and B2 is 0.25%, showing The kit of the present invention for detecting melon blight bacteria does have better sensitivity.

(C) Reproducibility test

In this experiment, a mixture containing 5% of contaminated seeds is used as the sample to be tested, and the primer pairs and probes shown in Table 1 are used for different interday and intraday variations. Coefficients of variation (CVs) analysis.

Table 3, the reproducibility test results of each group of samples to be tested in this experiment Group Different day analysis Different analysis on the same day B0 3.87±0.11% 8.35±5.18% B1 1.22±0.2% 1.3±0.15% B2 0.78±0.24% 1.5±0.37%

Please refer to Table 3, whether it is analyzed on different days or on the same day and at different times, the B1 and B2 groups have a lower coefficient of variation, which shows that the set of the present invention for detecting melon blight bacteria has a better reproduction Sex.

In summary, by using the primer pair and the probe in combination, the kit and method for detecting melon blight bacteria of the present invention can be applied to quickly and easily detect melon blight bacteria, and the detection results have high sensitivity and Reproducibility is the effect of the present invention.

Although the present invention has been disclosed using the above-mentioned preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with the art without departing from the spirit and scope of the present invention may make various changes and modifications relative to the above-mentioned embodiments. The technical scope of the invention is protected. Therefore, the scope of protection of the invention shall be subject to the scope of the attached patent application.

1: primer pair 11: Forward introduction 12: reverse primer 2: Probe 2a: Fluorescent marker 2a’: Falling off fluorescent marker 2b: Quench marks S1: Preparation steps of reaction mixture S2: lysis step S3: polymerization step S4: Repeat steps S5: Fluorescence detection step T: sample DNA T1: reverse single-stranded DNA T2: Forward single-stranded DNA

[Figure 1a] In the method for detecting melon wilt pathogen of the present invention, a schematic diagram of a lysis step S2. [Figure 1b] In the method for detecting melon wilt pathogen of the present invention, one of the schematic diagrams of a polymerization step S3. [Figure 1c] In the method of the present invention for detecting melon blight bacteria, the second schematic diagram of the polymerization step S3. [Figure 2] The flow chart of the method for detecting melon wilt pathogen of the present invention.

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1: primer pair

11: Forward introduction

12: reverse primer

2: Probe

2a: Fluorescent marker

2b: Quench marks

T: sample DNA

T1: reverse single-stranded DNA

T2: Forward single-stranded DNA

Claims (6)

A kit for detecting melon blight bacteria, which contains: A primer pair comprising a forward primer and a reverse primer, the forward primer has the nucleotide sequence shown in SEQ ID NO: 3, and the reverse primer has the nucleoside shown in SEQ ID NO: 4 Acid sequence; and A probe having the nucleotide sequence shown in SEQ ID NO: 7, one end of the probe has a fluorescent label, and the other end of the probe has a quenching label, and the quenching label is used to inhibit the The fluorescent marker generates a fluorescent signal. A kit for detecting melon blight bacteria, which contains: A primer pair comprising a forward primer and a reverse primer, the forward primer has the nucleotide sequence shown in SEQ ID NO: 5, and the reverse primer has the nucleoside shown in SEQ ID NO: 6 Acid sequence; and A probe having the nucleotide sequence shown in SEQ ID NO: 7, one end of the probe has a fluorescent label, and the other end of the probe has a quenching label, and the quenching label is used to inhibit the The fluorescent marker generates a fluorescent signal. As described in item 1 or 2 of the scope of patent application, the kit for detecting melon blight fungus, wherein the fluorescent marker is at least one selected from the group consisting of FAM, TET, NED and VIC. As described in item 1 or 2 of the scope of patent application, the kit for detecting melon blight fungus, wherein the quenching marker is selected from at least one of MGB, TAMRA and NFQ. As described in item 1 or 2 of the scope of the patent application, the kit for detecting melon blight fungus, wherein the 5'end of the probe has the fluorescent label FAM, and the 3'end of the probe has the quenching label MGB. A method for detecting melon blight bacteria, including: A reaction mixture preparation step is to mix a sample to be tested, a reaction buffer, a DNA mixture, the kit for detecting melon blight bacteria as described in item 1 or 2 of the scope of patent application, and A DNA polymerase to prepare a reaction mixture, the sample to be tested contains a sample of DNA; and A lysis step is to lyse the sample DNA in the reaction mixture into two single-stranded DNAs at 94-95°C, and the two single-stranded DNAs include a forward single-stranded DNA and a reverse single-stranded DNA; A polymerization step is to make the forward primer and the probe adhere to the reverse single-stranded DNA, and make the reverse primer adhere to the forward single-stranded DNA, and polymerize the DNA at 56-66°C The enzyme copies the sample DNA from the forward primer, the reverse primer and the probe, and at this time the fluorescent label of the probe falls off and releases a fluorescent signal; A repetition step is to sequentially repeat the lysis step and the polymerization step to amplify the sample DNA; and A fluorescent detection step is to detect the fluorescent signal released after the fluorescent mark of the probe falls off.

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