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Multi-RT-PCR (reverse transcription-polymerase chain reaction) method for simultaneously detecting citrus yellow vein clearing viruses and citrus tristeza viruses and application of multi-RT-PCR method

A technology of RT-PCR and decay virus, which is applied in the field of plant pathology, to avoid cross-contamination, save time and cost, and achieve authentic and credible results

Inactive Publication Date: 2017-03-08
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the simultaneous multiple PCR detection method of CYVCV and CTV in China. Therefore, a set of one-step multiple RT-PCR detection system for simultaneous detection of these two viruses is established to evaluate the infection of these two viruses in the field and in seedlings. The removal of virus during the detoxification process provides a fast, simple and reliable detection method

Method used

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  • Multi-RT-PCR (reverse transcription-polymerase chain reaction) method for simultaneously detecting citrus yellow vein clearing viruses and citrus tristeza viruses and application of multi-RT-PCR method
  • Multi-RT-PCR (reverse transcription-polymerase chain reaction) method for simultaneously detecting citrus yellow vein clearing viruses and citrus tristeza viruses and application of multi-RT-PCR method
  • Multi-RT-PCR (reverse transcription-polymerase chain reaction) method for simultaneously detecting citrus yellow vein clearing viruses and citrus tristeza viruses and application of multi-RT-PCR method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Test materials

[0039] The test material infected with two kinds of viruses, CTV and CYVCV, was Simmons sweet orange, which was provided by the National Citrus Seedling Virus Elimination Center of Citrus Research Institute, Chinese Academy of Agricultural Sciences. The leaves of this sweet orange were used as positive materials, and the leaves of healthy sweet orange seedlings were used as negative control materials. Field samples were randomly collected from 5-10 leaves in Beibei, Chongqing in September 2016.

[0040] 2. Total Nucleic Acid Extraction

[0041] Use the RNAiso Plus kit (TaKaRa, Japan) to extract the total nucleic acid of sweet orange leaves (collected directly from poisonous sources or healthy seedlings or stored in a -80°C refrigerator) according to the operating instructions, dissolve in 40 μL DEPC water, and detect the concentration with a spectrophotometer and purity, the ratio of A280 / A260 ranges from 1.8 to 2.2, and the extracted nucleic acid ...

Embodiment 2

[0047] Establishment of one-step multiplex RT-PCR system

[0048] Design specific primers, use the One Step RT-PCR Kit Ver.2 kit (TaKaRa, Japan), optimize and adjust the concentration of CTV and CYVCV primers that affect the one-step multiplex RT-PCR amplification, and compare the differences between the three pairs of primers. Concentration The final appropriate primer concentration was determined by the electrophoresis results of the PCR products. For the additional Mg added in the PCR system 2+ After optimizing the final concentration of dNTP, 7 concentration combinations (Table 2) were set, and the PCR product was subjected to electrophoresis to determine the best combination. According to the Tm values ​​of the three pairs of primers, the gradient temperature difference was set to 12°C for the annealing temperature by a gradient PCR instrument (BiometraTgradient), and a total of 12 annealing temperatures (T1-T12): 51.0, 51.3, 52.1, 53.4, 54.8, 56.3, 57.7, 59.1, 60.6, 61...

Embodiment 3

[0065] 1. One-step single and multiplex RT-PCR detection sensitivity comparison results

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Abstract

The invention relates to a multi-RT-PCR (reverse transcription-polymerase chain reaction) method for simultaneously detecting citrus yellow vein clearing viruses and citrus tristeza viruses. The multi-RT-PCR method has the advantages that Mg<2+> and dNTP (diethyl-nitrophenyl thiophosphate) concentration, primer concentration and annealing temperatures which can affect multi-PCR are adjusted and optimized by the aid of selected specific primers of conserved sequences and reference genes of coat proteins of two types of pathogens of the CYVCV (citrus yellow vein clearing viruses) and the CTV (citrus tristeza viruses), the one-step multi-RT-PCR method for simultaneously detecting the CYVCV and the CTV can be created, and the sensitivity of the multi-RT-PCR method is consistent with single RT-PCR detection sensitivity; as shown by results of detection on 33 field samples by the aid of the multi-RT-PCR method, the CYVCV and CTV infection rates are 54.5% and 66.7% respectively, and 36.4% of samples are infected in a mixed manner; the multi-RT-PCR method can be used for simultaneously quickly detecting the CYVCV and the CTV in large quantities of field samples and has important significance on improving efficient and accurate detection and timely and effective prevention and control on the citrus yellow vein clearing viruses and the citrus tristeza viruses.

Description

technical field [0001] The invention belongs to the field of plant pathology, and in particular relates to a multiple RT-PCR method for simultaneously detecting citrus yellow vein bright virus and decay virus and its application. Background technique [0002] Citrus yellow vein disease caused by Citrus yellow vein clearing virus (CYVCV) is a new disease in my country, which poses a serious threat to the citrus industry, especially the lemon industry. CYVCV is a positive-sense single-stranded RNA (+ssRNA) virus consisting of 7529 nucleotides, belonging to the genus Mandarivirus. In addition to CYVCV being transmitted by grafting and rubbing inoculation, it can also be transmitted by Spiraea aphid (Aphis spiraecola Patch) and bean aphid (A. Craccivora Koch) on kidney bean (Phaseolus vulgaris) and cowpea (Vignaunguiculata). Citrus tristeza virus (CTV), caused by Citrus tristeza virus (CTV), is a worldwide viral disease of citrus, causing serious economic losses to the citrus i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 刘金香赵恒燕周常勇陈洪明
Owner SOUTHWEST UNIVERSITY
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