Method for detecting southern rice black-streaked dwarf virus in sogatella furcifera mixed sample based on test strip and application
Through the detection method based on colloidal gold immunostrip strips, white-backed planthopper samples were screened and mixed, and the rapid detection of SRBSDV in mixed samples of white-backed planthoppers in the field was solved, and the problem of low detection efficiency of large sample sizes in the field in the prior art was solved, and efficient and economical disease monitoring and early warning was achieved.
Patent Information
- Application Number
- CN202510107403.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-23
- Publication Date
- 2025-04-18
AI Technical Summary
The existing technology is difficult to achieve rapid, economical and real-time detection of large samples of white-backed planthoppers in the field, resulting in inefficient monitoring and early warning of black rice dwarf shrinkage disease in southern rice.
The detection method based on colloidal gold immunostrip strips was adopted. By screening and mixing non-toxic white-backed planthoppers and poisonous white-backed planthoppers, mixed samples with 1-mixed 9 and 1-mixed 19 were formed. The colloidal gold test strips were used for detection, and the rapid detection of SRBSDV in mixed samples of white-backed planthoppers was achieved.
The detection cycle is shortened to 30-60 minutes, and the detection efficiency is increased by 10 times. It can detect large numbers of samples in real time and quickly in the field, achieving early accurate monitoring and early warning of diseases, and greatly reducing detection costs.
Smart Images
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Abstract
Description
Technical field:
[0001] The invention relates to a rapid detection method for southern rice black-streaked dwarf virus in a mixed sample of white-backed planthopper based on colloidal gold immunoassay paper strips, which can be applied to monitoring the growth and decline trend and epidemic law of southern rice black-streaked dwarf virus in rice fields in my country, and belongs to the field of agricultural science and technology. Background technology:
[0002] Southern rice black-streaked dwarf virus (SRBSDV) is a malignant rice virus disease caused by the spread of Southern rice black-streaked dwarf virus (SRBSDV) by white-backed planthoppers in a persistent and proliferative manner. Rice plants infected with SRBSDV are dwarfed, do not produce heading or produce necked headings, resulting in severe yield reduction or even total failure. In the eight years since it was discovered in Guangdong in 2001, its diseased area has expanded 100,000 times. In 2011 alone, the area of occurrence in my country reached 2 million hectares. It is currently one of the most serious rice diseases in southern rice-growing areas of my country. SRBSDV was detected in Jiangsu for two consecutive years in 2023 and 2024. Due to its great damage and difficulty in prevention and control, Southern rice black-streaked dwarf virus was included in the "List of Class I Crop Diseases and Pests" by the Ministry of Agriculture and Rural Affairs in 2020 and 2023, and it is the only viral disease among them.
[0003] Whether the white-backed planthopper is infected with the virus and the accurate infection rate are the core indicators for achieving early and accurate prediction of southern rice black-streaked dwarf disease, and all of these rely on the detection results of a large sample of white-backed planthoppers in the field. In view of the difficulties in purifying SRBSDV, the low titer and poor specificity of antibodies prepared with expressed proteins and synthetic peptides as immunogens, after multiple failed antibody trials, we broke through traditional thinking and pioneered a new "tumor excision immunity" method, successfully obtaining highly efficient and specific SRBSDV monoclonal antibodies. Based on the developed monoclonal antibodies, a dot immunobinding assay (DIBA) was established for the infection of single white-backed planthoppers. However, the DIBA method is suitable for detecting single white-backed planthoppers, and the detection cycle is 3-5 hours. Although molecular detection technologies based on PCR and other technologies have good specificity and high sensitivity, they can only be performed in laboratories and are not suitable for real-time rapid detection of large-scale field samples. The testing of a large number of samples over a large area requires a lot of manpower and material resources. Therefore, it is necessary to develop a fast, economical, and suitable detection technology for a large number of field samples to monitor the occurrence of southern rice black-streaked dwarf disease in sentinel points in key areas. When the "whistle" conditions are triggered, it is more economical and effective to start the next level of monitoring mechanism. Summary of the invention:
[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a rapid detection method for SRBSDV in a mixed sample of white-backed plant hoppers based on colloidal gold immunoassay strips, which is used to detect in real time whether the white-backed plant hopper population in the field is infected or not.
[0005] The present invention selects non-toxic white-backed planthoppers raised indoors, and highly toxic white-backed planthopper populations obtained after non-toxic white-backed planthoppers are fed and circulated on SRBSDV-infected rice plants as test materials. A rapid detection method for detecting SRBSDV in a mixed sample of white-backed planthoppers based on colloidal gold immunoassay strips is invented. Single-headed toxic white-backed planthoppers are screened out from 3-5-year-old white-backed planthopper nymphs or white-backed planthopper adults, and 9, 19, and 49 heads are taken out from the non-toxic white-backed planthopper population and mixed with the single-headed toxic white-backed planthoppers. 20ul of coating liquid is added to each white-backed planthopper and ground and mixed to form 1-mixed-9 and 1-mixed-19 white-backed planthopper mixed samples, respectively, that is, 1 out of 10 white-backed planthoppers carries SRBSDV, and so on. The colloidal gold test strips were used to test the mixed samples of white-backed planthoppers. The results showed that the mixed sample of 1 mixed with 9 white-backed planthoppers tested positive, and the mixed sample of 1 mixed with 19 tested negative, indicating that as long as 1 of 10 white-backed planthoppers carried SRBSDV, it could be detected. The detection cycle was shortened to 30-60 minutes, and the detection efficiency was increased by 10 times. The test results of the test strips of white-backed planthopper samples collected in the field were consistent with the results of the DIBA method and the RT-PCR method.
[0006] The mixed sample test strip detection method for white-backed planthoppers is suitable for real-time and rapid detection of large quantities of samples of different generations (first and second generations in rice fields) of white-backed planthoppers at SRBSDV monitoring sentinel points, realizing early and accurate monitoring and early warning of the disease. Whether in the intermittent period or the epidemic period of the disease, it can monitor the growth and decline trend and epidemic law of southern rice black-streaked dwarf disease in the field in real time, greatly reducing the detection cost and providing the necessary technical guarantee for the establishment of an accurate early warning and monitoring system for the disease. Description of the drawings:
[0007] Figure 1 : Results of the detection of white-backed planthopper populations (A, non-toxic white-backed planthopper population; B, white-backed planthopper population with 88% SRBSDV infection rate; C + , positive control; CK - , negative control).
[0008] Figure 2 : Screening results of single white-backed planthoppers carrying SRBSDV (A, DIBA method; B, RT-PCR method; M, DNA standard molecular weight; CK + , positive control; CK - , negative control).
[0009] Figure 3: Detection results of mixed samples of white-backed planthopper (A, test strip method; B, DIBA method; C, RT-PCR method; M, DNA standard molecular weight; 1, 1 mixed with 9; 2, 1 mixed with 19; CK + , positive control; CK - , negative control).
[0010] Figure 4 : Detection results of white-backed planthopper collected in the field (A, test strip method; B, DIBA method; 1, Yixing; 2, Jurong; CK + , positive control; CK - , negative control). Specific implementation method:
[0011] The methods used in the following examples are all conventional methods unless otherwise specified.
[0012] Example 1, SRBSDV test strips to detect mixed samples of white-backed planthoppers
[0013] Continuous rearing of white-backed planthoppers ( Figure 1 A), select 2nd-instar white-backed planthopper nymphs and feed them on rice plants carrying SRBSDV for 5 days. After the feeding, the white-backed planthoppers are moved to rice seedlings and fed for 8-10 days to pass the cycle period, and a white-backed planthopper population with a virus rate greater than 80% is obtained ( Figure 1 B).
[0014] 20 heads were randomly selected from the 3rd instar white-backed planthopper population carrying SRBSDV, and placed in 2mL centrifuge tubes. 20ul of coating solution was added, and the mixture was ground with a toothpick and allowed to stand for 5min. The single head of white-backed planthopper carrying SRBSDV was screened and tested. Figure 2 A), to further verify that the screened white-backed planthoppers carry SRBSDV, 6 white-backed planthoppers that tested positive by DIBA were randomly selected to extract RNA, and then tested and verified by RT-PCR ( Figure 2 B), the test results are consistent with those of the DIBA method.
[0015] Take 9 and 19 heads from the 3rd-instar non-toxic white-backed planthopper population and place them in the centrifuge tube of the single white-backed planthopper carrying SRBSDV obtained by the above screening. Add 20ul of coating solution to each white-backed planthopper, grind and mix, and form a 1-mixed 9 and 1-mixed 19 white-backed planthopper mixed sample, that is, 1 out of 10 white-backed planthoppers carries SRBSDV, and so on. Let it stand for 5 minutes, and draw 3 drops (about 150ul) of the white-backed planthopper mixed homogenate and add it to the sample well of the test strip. Observe the results after 5 minutes. If red strips appear on both the T line and the C line at the same time, it is positive. If red strips appear on the C line but not on the T line, it is negative. If red strips do not appear on both the C line and the T line, or if red strips appear only on the T line but not on the C line, the results are invalid.
[0016] The mixed samples of white-backed planthoppers (1 mixed with 9 and 1 mixed with 19) were tested and verified by DIBA method. Then, RNA was extracted from the mixed samples and verified again by RT-PCR.
[0017] The test strip test results showed that in the 1-to-9 white-backed planthopper mixed sample, red stripes appeared on both the T and C lines; while in the 1-to-19 white-backed planthopper mixed sample, no red stripes appeared on the T line, but red stripes appeared on the C line. This shows that the test strip can detect whether there are gray planthoppers carrying SRBSDV ( Figure 3 A). The results of DIBA and RT-PCR showed that the mixed samples of 1 mixed with 9 and 1 mixed with 19 were all positive for white-backed planthoppers ( Figure 3 B, 3C). The results showed that the test strips were reliable in detecting mixed samples of white-backed planthoppers. Their sensitivity was lower than that of DIBA and RT-PCR methods, but their detection efficiency was increased by 10 times. Moreover, they were suitable for real-time and rapid detection in the field, which greatly reduced the detection cost.
[0018] Example 2, detection of white-backed planthopper samples collected in the field
[0019] In May 2023, white-backed planthopper adults were collected from rice fields in Yixing and Jurong, Jiangsu Province. Ten white-backed planthoppers were randomly selected and tested with the test strips described in Example 1. The results showed that the Yixing sample was negative and the Jurong sample was positive ( Figure 4 ). Then 50 white-backed plant hoppers were randomly selected and tested by DIBA method. One of them was found to carry SRBSDV with a virus rate of 2%. The test result was consistent with the test result of mixed sample with test strips ( Figure 4 B).
[0020] The above embodiments do not limit the present invention in any form.
Claims
1. A method for detecting southern rice black-streaked dwarf virus in a mixed sample of white-backed planthoppers based on a test strip, characterized in that: The following steps are involved: (1) Mix 2-10 3-5-year-old white-backed planthopper nymphs or adults, add 20ul of coating solution to each white-backed planthopper, grind and mix well; (2) After standing for 5 min, 150 μl of the supernatant was added to the test well of the test strip; (3) After standing for 5 minutes, observe the results. If red stripes appear on both the C line and the T line, at least one of the white-backed planthoppers tested is carrying SRBSDV. If red stripes appear on the C line but not on the T line, then none of the white-backed planthoppers tested is carrying SRBSDV.
2. Application of a detection method for detecting SRBSDV in a mixed sample of white-backed plant hoppers based on a test strip according to claim 1 in detecting whether a white-backed plant hopper population is infected or not.