Method for detecting rice stripe virus in laodelphax striatellus mixed sample based on test strip and application

Abstract

The invention provides a rapid detection method for detecting a rice stripe virus in a laodelphax striatellus mixed sample based on a colloidal gold immune test strip. The method is used for detecting whether laodelphax striatellus carrying RSV exists in a 3-5-age laodelphax striatellus nymph or adult population, and only one laodelphax striatellus carrying RSV within 2-50 laodelphax striatellus can be detected. The detection period is shortened to 30-60 minutes, the detection method is simple, convenient and rapid, low in cost and suitable for field real-time detection, and necessary technical guarantee is provided for establishment of a precise early warning and monitoring system for the rice stripe disease.

Description

Technical field:

[0001] The invention relates to a rapid detection method for rice stripe virus in a mixed sample of gray planthopper based on colloidal gold immunoassay paper strips, which can be applied to monitoring the growth and decline trend and epidemic law of rice stripe leaf blight in the field and belongs to the field of agricultural science and technology. Background technology:

[0002] Rice stripe leaf blight is a rice virus disease caused by the transmission of Rice stripe virus (RSV) by the Laodelphax striatellus in a persistent trans-oviposit manner. It is one of the most serious rice diseases in rice-wheat rotation areas since the 20th century. Infection of rice can cause the entire plant to die, prevent normal heading, and severely reduce yields. It is called the "cancer" of rice. Since 2000, rice stripe leaf blight has been widely prevalent in the japonica rice planting areas in the lower reaches of the Yangtze River in my country. Generally, the yield of the field is reduced by 20%-30%, and the incidence rate of serious fields is as high as 80%-90%. Some fields even have no harvest, posing a serious threat to the safe production of japonica rice in my country.

[0003] In recent years, with the large-scale promotion of rice stripe disease-resistant varieties and the application of scientific and green control technology for field gray leaf hoppers, the rapid coverage of disease-resistant varieties in southern japonica rice areas has been achieved, the disease has been effectively controlled in my country's main rice-producing areas, and the disease has entered an intermittent period. However, given that the epidemic of viral diseases involves multiple factors such as hosts, viruses, vectors, and the environment, it has the characteristics of intermittent outbreaks, indicating that there is a risk of another outbreak of rice stripe disease. Monitoring and early warning of it is a long-term and important task. Whether the vector gray leaf hopper is infected with the virus and its infection rate are the core indicators for early warning and accurate prediction of rice stripe disease, and all of these rely on the detection results of a large sample of gray leaf hoppers in the field. In the early stage, a dot immunobinding assay (DIBA) was created based on RSV monoclonal antibodies to detect whether a single gray leaf hopper carries RSV and obtain the infection rate of the gray leaf hopper population. This detection technology has been widely promoted and applied in grassroots agricultural technical departments and has been listed as the only industry standard for the detection of virus-carrying gray leafhoppers (NY / T 2385-2013). However, the DIBA method is suitable for detecting single gray leafhoppers, and the detection cycle is 3-5 hours. Molecular detection technologies based on PCR, etc., although with good specificity and high sensitivity, can only be performed in laboratories and are not suitable for real-time rapid detection of large-scale field samples. At present, rice stripe disease is in the inter-epidemic period in my country, and comprehensive detection of large areas of gray leafhoppers and large samples wastes manpower and material resources. Therefore, it is necessary to develop real-time, rapid, and suitable technology for the detection of large numbers of gray leafhoppers in the field, regularly monitor the growth and decline trends of rice stripe disease in the field at disease warning sentinel points in key areas, and activate the next level of monitoring and early warning mechanism when the "whistle" condition is triggered, which is more in line with the current reasonable response mechanism to the epidemic law of the disease. 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 detecting RSV in mixed samples of gray leaf hoppers based on colloidal gold immunoassay strips, which is used to detect in real time whether the gray leaf hopper population in the field is infected or not.

[0005] The present invention selects non-toxic gray planthoppers raised indoors and gray planthopper groups with an RSV infection rate of more than 95% as test materials. A rapid detection method for detecting RSV in a gray planthopper mixed sample based on colloidal gold immunoassay strips is invented. Single-headed gray planthoppers with virus are screened out from a group of 3-5-year-old gray planthopper nymphs or adults, and 9, 19, 49, and 59 are taken out from the non-toxic gray planthopper group and mixed with the single-headed gray planthopper with virus. 20ul of coating liquid is added to each gray planthopper and ground and mixed to form gray planthopper mixed samples of 1 mixed with 9, 1 mixed with 19, 1 mixed with 49, and 1 mixed with 59, respectively, that is, 1 out of 10 gray planthoppers carries RSV, and so on. The colloidal gold test strips were used to test the mixed samples of gray leafhoppers. The results showed that the mixed samples of gray leafhoppers 1 mixed with 9, 1 mixed with 19, and 1 mixed with 49 tested positive, and the test result of 1 mixed with 59 was negative, indicating that as long as one of the 50 gray leafhoppers carried RSV, it could be detected. The detection cycle was shortened to 30-60 minutes, and the detection efficiency was increased by 50 times. The test results of the gray leafhopper sample strips 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 of gray planthopper is suitable for real-time and rapid detection of large quantities of samples of different generations (overwintering generation, first generation in rice fields and second generation) of gray planthopper populations in the field at the rice stripe disease 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, the growth and decline trend and epidemic law of rice stripe disease in the field can be monitored 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 detection of Laodelphax striatellus population (A, Laodelphax striatellus population without RSV infection; B, Laodelphax striatellus population with RSV infection rate of 98%; C + , positive control; CK - , negative control).

[0008] Figure 2 : Screening results of single-headed Laodelphax striatellus carrying RSV (A, DIBA method; B, RT-PCR method; M, DNA standard molecular weight; CK + , positive control; CK - , negative control).

[0009] Figure 3: Results of mixed samples of Laodelphax striatellus (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; 3, 1 mixed with 49; 4, 1 mixed with 59; CK + , positive control; CK - , negative control).

[0010] Figure 4 : Results of field collection of Laodelphax striatellus (A, test strip method; B, DIBA method; 1, Yixing; 2, Dafeng; 3, Tongzhou; 4, Jiangyan; 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, RSV test strips to detect mixed samples of Laodelphax striatellus

[0013] After mating, the adult gray leafhoppers were reared on rice seedlings in the insectary room, and a single female insect laid eggs alone. After laying eggs, the female insects were tested for RSV. The offspring of the non-toxic female insects and the offspring of the infected female insects were reared separately. After three generations of screening and rearing, the non-toxic gray leafhopper population was obtained. The insects were reared continuously in the insectary room, and the insects were taken regularly to test whether they were contaminated by RSV ( Figure 1 A). The offspring of each infected female insect are raised together, mated in groups, and the infection rate of the group is tested after they lay eggs. Only the offspring groups with an infection rate of more than 90% are retained for further breeding. After three generations of screening, the remaining offspring groups are mixed and raised to obtain a group of gray leafhoppers with an infection rate greater than 90% ( Figure 1 B).

[0014] 20 heads were randomly selected from the 3rd instar population of RSV-carrying gray leafhoppers, placed in 2mL centrifuge tubes, 20ul of coating solution was added, ground with a toothpick and mixed, and allowed to stand for 5min. The single head of virus-carrying gray leafhoppers ( Figure 2 A), to further verify that the selected gray leafhoppers carry RSV, 6 gray leafhoppers 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, 19, 49 and 59 heads from the 3rd-instar non-toxic gray planthopper population and place them in the centrifuge tube of the single gray planthopper carrying RSV obtained by the above screening. Add 20ul of coating solution to each gray planthopper, grind and mix, and form mixed samples of gray planthoppers of 1 mixed with 9, 1 mixed with 19, 1 mixed with 49 and 1 mixed with 59, that is, 1 out of 10 gray planthoppers carries RSV, and so on. Let it stand for 5 minutes, and take 3 drops (about 150ul) of the gray planthopper mixed homogenate and add it to the sample well of the test strip. Observe the results after 5 minutes. If red stripes appear on both the T line and the C line at the same time, it is positive. If red stripes appear on the C line but not on the T line, it is negative. If red stripes appear on both the C line and the T line, or if red stripes appear only on the T line but not on the C line, the result is invalid.

[0016] Mixed samples of Laodelphax striatellus from 1-mixed 9, 1-mixed 19, 1-mixed 49 and 1-mixed 59 were tested and verified by DIBA method. Then, RNA of the samples was extracted and verified again by RT-PCR.

[0017] The test results showed that the mixed samples of 1 mixed with 9, 1 mixed with 19 and 1 mixed with 49 showed red bands on both the T and C lines; while the mixed sample of 1 mixed with 59 showed no red bands on the T line but a red band on the C line. This indicates that the test strip can detect whether there are 50 LAPHs carrying RSV ( Figure 3 A). The results of DIBA and RT-PCR showed that the mixed samples of 1-mixed-9, 1-mixed-19, 1-mixed-49 and 1-mixed-59 were all positive for Laodelphax striatellus ( Figure 3 B, 3C). The results showed that the test strips were reliable in detecting mixed samples of Laodelphax striatellus. Their sensitivity was lower than that of DIBA and RT-PCR methods, but their detection efficiency was increased by 50 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 samples of Laodelphax striatellus collected in the field

[0019] In May 2023, adult gray leaf hoppers were collected from rice fields in Yixing, Dafeng, Tongzhou and Jiangyan, Jiangsu Province. Fifty gray leaf hoppers were randomly selected and tested with the test strips described in Example 1. The results were all negative ( Figure 4 A). Then 50 gray leaf hoppers were randomly selected and tested by DIBA method. The virus carrying rate of a single gray leaf hopper was 0. The test result was consistent with the test result of mixed sample with test strip ( Figure 4 B).

[0020] The above embodiments do not limit the present invention in any form.

Claims

1. A method for detecting rice stripe virus in a mixed sample of Laodelphax striatellus based on a test strip, characterized in that: The following steps are involved: (1) 2-50 3-5th-instar nymphs or adults of Laodelphax striatellus are mixed, and 20 μl of coating solution is added to each Laodelphax striatellus, and the mixture is ground and mixed evenly; (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 gray leafhoppers tested is carrying RSV. If red stripes appear on the C line but not on the T line, none of the gray leafhoppers tested is carrying RSV.

2. Use of the method for detecting rice stripe virus in a mixed sample of gray leaf hopper based on a test strip according to claim 1 in detecting whether a gray leaf hopper population is infected with the virus.

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