AU2020103434A4 - Primers and kit for detecting four kiwifruit viruses of acva, acvb, cmv and accrav - Google Patents

Primers and kit for detecting four kiwifruit viruses of acva, acvb, cmv and accrav Download PDF

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AU2020103434A4
AU2020103434A4 AU2020103434A AU2020103434A AU2020103434A4 AU 2020103434 A4 AU2020103434 A4 AU 2020103434A4 AU 2020103434 A AU2020103434 A AU 2020103434A AU 2020103434 A AU2020103434 A AU 2020103434A AU 2020103434 A4 AU2020103434 A4 AU 2020103434A4
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primers
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acvb
cmv
accrav
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Jing Shang
Min Zhang
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    • C12Q1/701—Specific hybridization probes
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

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Abstract

The present invention discloses primers and a kit for detecting four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV. The primers includes a system I and a system II used in combination; at least one of the system I and system II includes primers as shown in SEQ ID NO.1-2 and SEQ ID NO.3-4. The system I and system II also include primers as shown in SEQ ID NO.5-6 or primers as shown in SEQ ID NO.7-8, and the system I and system II include different primers. The present invention can effectively detect the four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV simultaneously by designing two sets of primer systems, and can avoid mutual interference between primers.

Description

PRIMERS AND KIT FOR DETECTING FOUR KIWIFRUIT VIRUSES OF ACVA, ACVB, CMV AND ACCRAV TECHNICAL FIELD
The present invention relates to the technical field of genetic engineering, and in particular, to
primers, a kit and a detection method for detecting four kiwifruit viruses of AcVA, AcVB, CMV and
AcCRaV.
BACKGROUND
Plant viruses are intracellular parasitic pathogens composed of nucleic acids and proteins with
infective activity. According to the eighth report of the International Committee on Taxonomy of
Viruses (ICTV), there are currently 1,122 plant virus species found worldwide. Plant diseases
caused by plant viruses are known as "plant cancer", and their damage to crops is only secondary to
fungal pathogens. Almost every crop is harmed by 2-3 viruses. The annual loss caused by plant
viruses is about $40 billion around the world, and the damage caused by tobacco mosaic virus
(TMV) alone is more than $100 million.
Kiwifruit (Actinidia spp.) is one of the most important horticultural crops in the world. Owing
to the rich content of vitamin C, minerals and dietary fiber in the pulp, kiwifruit is favored by the
public. In 2017, the world's kiwifruit yield reached 403 million tons, but the yield and quality of
kiwifruit, even the healthy development of kiwifruit industry are affected by diseases infection.
Virus disease is a kind of disease with long latent period and great hidden danger in production. The
viruses Actinidia chlorotic ringspots-associatedvirus (AcCRaV), Cucumber mosaic virus (CMV),
Actinidia virus A (AcVA) and Actinidia virus B (AcVB) can infect kiwifruit under natural
conditions, which has certain influence on the normal growth and development of fruit trees and
fruits of kiwifruit. Additionally, these viruses are detected in Chinese kiwifruit cultivars in Hubei,
Shaanxi and Sichuan provinces. Currently, however, there is no report that the four viruses can be
detected simultaneously without mutual interference between the primers.
SUMMARY
In view of the above-mentioned deficiencies in the prior art, the present invention provides
primers and a kit for detecting four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV, which
can detect the four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV, and the primers have
good specificity and high sensitivity.
In order to achieve the above-mentioned objective, the technical solution adopted by the
present invention to solve the technical problem is as follows.
Primers for detecting four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV, including a
system I and a system II used in combination; at least one of the system I and the systemII includes
primers as shown in SEQ ID NO.1-2 and SEQ ID NO.3-4.
The system I and the system II also include primers as shown in SEQ ID NO.5-6 or primers as
shown in SEQ ID NO.7-8; the primers shown in SEQ ID NO.5-6 and the primers shown in SEQ ID
NO.7-8 do not simultaneously appear in the system I and the system II; namely, when the system I
includes the primers shown in SEQ ID NO.5-6, the system II does not include the primers shown in
SEQ ID NO.5-6, but includes the primers shown in SEQ ID NO.7-8.
The specific sequences of the primers are as follows:
AcVB-F: 5'-GTTTGCGAGGAGACGTAGGGC-3'(SEQ ID NO.1);
AcVB-R: 5'-AGTTAAGTGCTCTYGGRGGTGTG-3'(SEQ ID NO.2);
CMV-F: 5'-GCCGCCATCTCTGCTATGTT-3'(SEQ ID NO.3);
CMV-R: 5'-GAATGCGTTGGTGCTCGATG-3'(SEQ ID NO.4);
AcVA-F: 5'-CATGGCAAAGAATATCTCAAG-3'(SEQ ID NO.5);
AcVA -R: 5'-AGATCCAACCCAGAGTTGAAA-3'(SEQ ID NO.6);
AcCRaV-F: 5'-GCTTGCAAGATGTCGATGCAG-3'(SEQ ID NO.7);
AcCRaV-R: 5'-TGCAGGGTCTGCTGCTTATTA-3'(SEQ ID NO.8).
Further, the system I includes primers as shown in SEQ ID NO.1-6; the system II includes
primers as shown in SEQ ID NO.1-4 and the primers as shown in SEQ ID NO.7-8.
Further, the system I includes the primers as shown in SEQ ID NO.1-6; the system II includes
the primers as shown in SEQ ID NO.7-8.
Further, the system I includes the primers as shown in SEQ ID NO.5-6; the system II includes
the primers as shown in SEQ ID NO.1-4 and the primers as shown in SEQ ID NO.7-8.
A method for detecting the four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV,
including the following steps:
(1) collecting an infected sample and extracting a total RNA of the infected sample to obtain
cDNA by reverse transcription (RT);
(2) performing a polymerase chain reaction (PCR) with the primers of the system I and the
primers of the system II in claim 1, respectively.
Further, after collecting the infected sample, selecting a corresponding primer system of the
system I or the system II for a preliminary detection according to the symptoms shown by the
sample, and then using the primers of the residual system for a supplementary detection.
Further, a PCR system includes: 12.5 L of 2xTaq PCR Master Mix, 1.0 L of cDNA, 1.0 L
of the primers of the system I or the primers of the systemII, and supplementary ddH 2 0 to 25 L.
Further, each of the primers of system I is added with an equal proportion concentration; a
concentration of the primers shown in SEQ ID NO.1-2 of the system II is 5 times of that of other
primers, and other primers are added with an equal proportion concentration.
Further, a procedure of the PCR is as follows: an initial denaturation at 94°C for 3 min;
followed by 35 cycles of a denaturation at 94°C for 30 s, an annealing at 54°C for 30 s, an extension
at 72°C for 40 s; and an extension at 72°C for 10 min.
A kit for detecting the four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV, including
the primers in claim 1.
The advantages of the present invention are as follows.
The present invention can effectively detect four kiwifruit viruses of AcVA, AcVB, CMV and
AcCRaV simultaneously by designing two sets of primer systems, and can avoid mutual
interference between primers, and the primers also have high sensitivity.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram showing results of a specific detection of different primers;
FIG. 2 is a diagram showing results of a sensitivity detection of a primer system of AcVA-F/R,
AcVB-F/R and CMV-F/R;
FIG. 3 is a diagram showing results of a sensitivity detection of a primer system of AcVB-F/R,
CMV-F/R and AcCRaV-F/R;
FIG. 4 is a diagram showing results of a specific detection of four viruses by the primer system
of AcVA-F/R, AcVB-F/R and CMV-F/R, and the primer system of AcVB-F/R, CMV-F/R and
AcCRaV-F/R.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The specific embodiment of the present invention are described below to facilitate those
skilled in the art to understand the present invention. However, it should be understood that the
present invention is not limited to the scope of specific embodiment. For ordinary technical
personnel in the art, these variations are obvious as long as various variations are within the spirit
and scope of the present invention defined and determined by claims, and all inventions and
creations utilizing the concepts of the present invention are intended to be protected.
Embodiment
1. Sample collection
Leaves with typical symptoms of vesicular contraction, ring spots, yellow spots, wilting
yellow spots, long and narrow deformation, yellow vein and necrosis are collected from kiwifruit
planting areas in Sichuan Province. Leaf samples are stored with anhydrous calcium chloride in a
dry bottle at 4°C. Fresh leaf samples are stored in a refrigerator at -80°C.
2. Total RNA extraction
Plant tissue samples (0.1 g) are ground in liquid nitrogen with 2 ml sterilized centrifuge tubes,
extracted with phenol, chloroform, isoamyl alcohol (v: v: v, 25: 24: 1) (500 L) and an RNA buffer
(500 pL). After centrifugation, a supernatant (about 400 L) is mixed with 2 times the volume of 4
M lithium chloride solution, incubated at -20°C for at least 8 h, and then centrifuged at 4°C, 12,000
rpm for 10 min to recover RNA from particles. Pellets are centrifuged repeatedly with 70% ethanol
at 4°C, dried, and then suspended in 30 1 of ddH20 and stored at -20°C.
3. Primer design
Virus-specific primers are designed based on the conserved regions of each viral coat protein
(CP) gene obtained from GenBank using DNAMAN 8.0 and MEGA 7.0 software. The specificity
of the primers is verified on NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using BLASTN
tool. All of the primers are synthesized by Sangon Biotech Co., Ltd (Shanghai, China), and diluted
to 10 M and 50 M with ddH 20 for PCR amplification.
4. Specific detection of primers
RNA of AcVA, AcVB, CMV and AcCRaV is used as a template respectively for reverse
transcription to obtain cDNA of a corresponding single virus. The reverse transcription kit is
purchased from Sangon Biotech Co., Ltd (Shanghai, China).
A reverse transcription system includes: 4 L of the RNA; 2 L of a downstream primer, 2 L
of 5xRT buffer, 1 L of dNTP mixture, 0.2 L of RNase inhibitor, 0.2 L of M-mulv, and
supplementary RNase-free water to 10 L.
The reverse transcription system is placed in a water bath at 72°C for 5 min for reverse
transcription, followed by placed at 4°C for 5 min to obtain cDNA of each single virus.
Then the single cDNA of the AcVA, AcVB, CMV and AcCRaV is used as a template to
perform PCR amplification on the AcVA, AcVB, CMV and AcCRaV using the specific primers of
different virus shown in SEQ ID NO.1-8.
A reaction system includes: 12.5 L of 2xTaq PCR Master Mix, 1.0 L viral cDNA, 1.0 L of
each of upstream primers and downstream primers, and supplementary ddH2 0 to 25 L.
A reaction procedure is as follows: an initial denaturation at 94°C for 3 min; followed by 35
cycles of a denaturation at 94°C for 30 s, an annealing at 54°C for 30 s, an extension at 72°C for 40 s;
and an extension at 72°C for 10 min.
Finally, amplified products are analyzed by electrophoresis in 1% agarose gels, and the results
are shown in FIG. 1. In FIG. 1, lane M is Marker, lane 1 is AcVA, lane 2 is AcVB, lane 3 is CMV,
and lane 4 is AcCRaV. According to the detection results in FIG. 1, the electrophoresis lanes of
different virus are clearly visible, indicating that the primers corresponding to different virus have
strong specificity and sensitivity.
5. Establishment of multiplex PCR
(1) The primers in the reverse transcription system in step 4 are replaced by a mixed system of
AcVA-R, AcVB-R and CMV-R (c: c: c, 1: 1: 1), or a mixed system of AcVB-R, CMV-R and
AcCRaV-R (c: c: c, 5: 1: 1), and other processes are the same as those in step 4.
(2) The primers in the PCR system in step 4 are replaced by a mixed system of AcVA-F/R,
AcVB-F/R and CMV-F/R (c: c: c, 1: 1: 1), or a mixed system of AcVB-F/R, CMV-F/R and
AcCRaV-F/R R (c: c: c, 5: 1: 1), and the other processes are the same as those in step 4.
6. Sensitivity detection of multiplex PCR
The concentration of the total RNA extracted in step 2 is adjusted to 600 ng/L, then reverse transcribed with the mixed system of the primers established in step 5 to obtain cDNA. The cDNA is diluted by 10-fold continuous gradient (10°-10-8) as a template for PCR amplification, and then amplified by PCR using the PCR amplification primer system established in step 5. The sensitivity is analyzed by electrophoresis in 1% agarose gels, and the results are shown in FIG. 2, FIG. 3 and
FIG. 4.
FIG. 2 is a diagram showing results of a sensitivity detection of a triple primer system of
AcVA-F/R, AcVB-F/R and CMV-F/R.
FIG. 3 is a diagram showing results of a sensitivity detection of a triple primer system of
AcVB-F/R, CMV-F/R and AcCRaV-F/R.
Lanes 1 are 600 ng/4L; lanes 2 are 600x10' ng/L; lanes 3 are 600x10-2 ng/L; lanes 4 are
600x10-3 ng/L; lanes 5 are 600x10-4 ng/4L; lanes 6 are 600x10-5 ng/L; lanes 7 are 600x10-6
ng/4L; lanes 8 are 600x10-7 ng/4L; lanes 9 are 600x10 -8 ng/L, lanes M are 2000 bp molecular
markers; and lanes N are negative controls in both FIG. 2 and FIG. 3.
According to the detection results in FIG. 2 and FIG. 3, the primer system of AcVA-F/R,
AcVB-F/R and CMV-F/R, and the primer system of AcVB-F/R, CMV-F/R and AcCRaV-F/R have
high sensitivity.
FIG. 4 is a diagram showing results of a specific detection of four viruses by the primer system
of AcVA-F/R, AcVB-F/R and CMV-F/R, and the primer system of AcVB-F/R, CMV-F/R and
AcCRaV-F/R.
In FIG. 4, lane 1 is AcVA, lane 2 is AcVB, lane 3 is CMV, lane 4 is AcCRaV, lane 5 is AcVA + AcVB, lane 6 is AcVA + CMV, lane 7 is AcVB + CMV, lane 8 is AcVB+AcCRaV, lane 9 is CMV +
AcCRaV, lane 10 is AcVA + AcVB + CMV, lane 11 is AcVB + CMV + AcCRaV, lane M is a 2000
bp molecular marker, and lane N is a negative control.
According to the detection results in FIG. 4, the primer system of AcVA-F/R, AcVB-F/R and
CMV-F/R, and the primer system of AcVB-F/R, CMV-F/R and AcCRaV-F/R have high sensitivity.

Claims (10)

CLAIMS WHAT IS CLAIMED IS:
1. Primers for detecting four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV, comprising
a system I and a system II used in combination; wherein at least one of the system I and the system
II comprises primers as shown in SEQ ID NO.1-2 and SEQ ID NO.3-4;
the system I and the system II also comprise primers as shown in SEQ ID NO.5-6 or primers
as shown in SEQ ID NO.7-8; the primers shown in SEQ ID NO.5-6 and the primers shown in SEQ
ID NO.7-8 do not simultaneously appear in the system I and the system II.
2. The primers for detecting the four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV
according to claim 1, wherein, the system I comprises primers as shown in SEQ ID NO.1-6; the
system II comprises primers as shown in SEQ ID NO.1-4 and the primers as shown in SEQ ID
NO.7-8.
3. The primers for detecting the four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV
according to claim 1, wherein, the system I comprises the primers as shown in SEQ ID NO.1-6; the
system II comprises the primers as shown in SEQ ID NO.7-8.
4. The primers for detecting the four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV
according to claim 1, wherein, the system I comprises the primers as shown in SEQ ID NO.5-6; the
system II comprises the primers as shown in SEQ ID NO.1-4 and the primers as shown in SEQ ID
NO.7-8.
5. A method for detecting four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV,
comprising the following steps:
(1) collecting an infected sample and extracting a total RNA of the infected sample to obtain
cDNA by reverse transcription (RT);
(2) performing a polymerase chain reaction (PCR) with the primers of the system I and the
primers of the system II in claim 1, respectively.
6. The method for detecting the four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV
according to claim 5, wherein, after collecting the infected sample, selecting a corresponding primer
system of the system I or the system II for a preliminary detection according to the symptoms
shown by the sample, and then using the primers of the residual system for a supplementary
detection.
7. The method for detecting the four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV
according to claim 5, wherein, a PCR system comprises: 12.5 L of 2xTaq PCR Master Mix, 1.0 L
of cDNA, 1.0 L of the primers of the system I or the primers of the systemII, and supplementary
ddH 20 to 25 L.
8. The method for detecting the four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV
according to claim 7, wherein, each of the primers of system I is added with an equal proportion
concentration; a concentration of the primers shown in SEQ ID NO.1-2 of the system II is 5 times
of that of other primers, and other primers are added with an equal proportion concentration.
9. The method for detecting the four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV
according to claim 5, wherein, a procedure of the PCR is as follows: an initial denaturation at 94°C
for 3 min; followed by 35 cycles of a denaturation at 94°C for 30 s, an annealing at 54°C for 30 s, an
extension at 72°C for 40 s; and an extension at 72°C for 10 min.
10. A kit for detecting four kiwifruit viruses of AcVA, AcVB, CMV and AcCRaV, comprising
the primers according to claim 1.
AU2020103434A 2020-04-23 2020-11-13 Primers and kit for detecting four kiwifruit viruses of acva, acvb, cmv and accrav Ceased AU2020103434A4 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111363853A (en) * 2020-04-23 2020-07-03 四川农业大学 Primer and kit for detecting four kiwi fruit viruses of AcVA, AcVB, CMV and AcCRaV

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104762409A (en) * 2015-04-30 2015-07-08 四川农业大学 Method for detecting pseudomonas syringaepv altinidia through recombinase-mediated isothermal amplification technology
CN109628604A (en) * 2018-12-05 2019-04-16 中国农业科学院北京畜牧兽医研究所 New circRNA relevant to intramuscular fat and its application
CN110295255B (en) * 2019-08-16 2021-07-27 四川大学 RT-LAMP-LFD-based rapid detection method for detecting kiwi chlorotic and ringspot related viruses
CN111363853B (en) * 2020-04-23 2022-07-29 四川农业大学 Primer and kit for detecting four kiwi fruit viruses of AcVA, AcVB, CMV and AcCRaV

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111363853A (en) * 2020-04-23 2020-07-03 四川农业大学 Primer and kit for detecting four kiwi fruit viruses of AcVA, AcVB, CMV and AcCRaV
CN111363853B (en) * 2020-04-23 2022-07-29 四川农业大学 Primer and kit for detecting four kiwi fruit viruses of AcVA, AcVB, CMV and AcCRaV

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