A method for identifying mealybugs of Lycoris radiata and its application

Abstract

The invention discloses a method for identifying Phenacoccus solani, and relates to the field of molecular biology. A target for molecular biological identification of Phenacoccus solani, the target is a single sequence target or a target combination consisting of two sequences, the nucleotide sequence of the single sequence target is shown in SEQ ID No.1; the nucleotide sequence of the target combination is shown in SEQ ID No.1 and SEQ ID No.13. The invention provides a method for identifying Phenacoccus solani, which is identified by multi-channel fluorescence quantitative PCR, can improve the accuracy of the detection result, and shorten the detection time.

Description

Technical Field

[0001] The invention relates to the field of molecular biology, and in particular to a method for identifying powdery mildew larvae and an application thereof. Background Art

[0002] Lycoris mealybug (Phenacoccus solani) belongs to the Pseudococcidae family (Phenacoccus) of the order Hemiptera. The pest was first discovered in 1908 on mallow (Malva sinensis Cavan.) in California, USA. With the increase in international trade, it has now spread to all continents except Antarctica. In 1999, the insect was first discovered in Taiwan, my country; in 2008, the insect was discovered in Xinjiang and Beijing, and in recent years it has occurred in parts of central and southern my country. Lycoris mealybug has a wide diet, with hosts from as many as 40 families and 101 genera, involving more than 131 plant species. It especially likes to eat economic crops such as Solanaceae, Asteraceae and succulents.

[0003] The insect is tiny, and mainly harms host plants with female adults and nymphs. It harms the young and tender parts of the host plants, such as young branches, leaves, flower buds and petioles, by sucking the juice, which causes the growth of the damaged plants to weaken and even lead to a total failure in severe cases. At the same time, the honeydew secreted induces sooty mold, which hinders the photosynthesis of the plants, thus causing poor growth of the branches and leaves of the host plants and reducing the yield. According to the results of the risk analysis of the potential danger of the introduction of Lycoris radiata mealybug into my country, it is shown that the pest has a wide range of suitable habitats in my country. If it spreads widely as expected, it will cause great harm to the potato and vegetable crop production areas in the middle and lower reaches of the Yellow River and the Yangtze River, and is identified as a high-risk pest. The insect is also listed as an entry quarantine pest by Japan and South Korea.

[0004] Lycoris mealybug is a small insect with different sexes. The female adult is oval and covered with white wax powder. It can reproduce parthenogenetically or sexually. Its main identification characteristics are: the body is broadly oval, 23.2-27.1mm long and 1.3-1.6mm wide. Each trochanter has two sensory holes, and the tibia of the hind leg has many transparent holes. There are 6 bristles around the anal ring. The last pair of thorns has 2 cones and 10-13 trigonal glands, and the other thorns have 2 cones and 6 trigonal glands; the tail valve is well developed. The female adult lays eggs in the cotton-like egg sac near the genital pore. The newly hatched nymphs are active and can feed and cause harm within a short time after crawling out of the egg sac. Lycoris mealybug has a very strong reproductive capacity, the adult lays a large number of eggs, the population overlaps generations, and the population can grow at a very fast rate.

[0005] The body surface of Lycoris radiata mealybugs is often covered with white wax. During morphological identification, it is usually necessary to dissect the female adult and make slides to observe the various ductal gland characteristics on the insect cuticle for identification. The process is complicated and time-consuming. Especially when encountering closely related species, missing identification feature parts or non-adult stage samples, it is difficult to carry out traditional morphological identification methods.

[0006] At present, the methods used for identification of mealybugs in Lycoris radiata mainly include: 1) morphological characteristics identification; 2) PCR technology, etc. Among them, morphological characteristics identification requires a complete insect body for experiment, and the identification process is relatively complicated and has certain technical difficulties; PCR technology classifies and identifies mealybugs through DNA sequences, and the results are accurate, but the sequencing takes a long time. Therefore, in order to prevent the introduction of mealybugs in Lycoris radiata, a rapid, highly sensitive and highly specific identification and detection method for the insect of the present invention has important social and economic significance. Using the detection method of the present invention, mealybugs in Lycoris radiata can be effectively detected and the detection rate can be improved; it is helpful to carry out targeted prevention of the invasion and spread of mealybugs in Lycoris radiata, and protect the safety of agricultural production and ecological security in my country. Summary of the invention

[0007] In order to solve the deficiencies in the prior art, the present invention provides a method for identifying mealybugs on Lycoris radiata and its application.

[0008] The invention provides a target for molecular biological identification of Phenacoccus solani. The target is a target of a single sequence or a target combination consisting of two sequences. The nucleotide sequence of the target of the single sequence is shown as SEQ ID No.1; the nucleotide sequence of the target combination is shown as SEQ ID No.1 and SEQ ID No.13.

[0009] The invention provides application of the target in identifying Lycoris radiata powderycosa.

[0010] The present invention provides a primer combination for identifying Lycoris radiata mealybugs.

[0011] The primer combination includes a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID No. 2, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 3;

[0012] Alternatively, the primer combination includes three forward primers and four reverse primers, the nucleotide sequence of the forward primer combination is shown in SEQ ID No.2, SEQ ID No.5 and SEQ ID No.6, and the nucleotide sequence of the reverse primer combination is shown in SEQ ID No.3, SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9.

[0013] The present invention provides a probe for identifying Lycoris radiata mealybugs.

[0014] When the primer combination described above is used, and the primer combination includes a forward primer and a reverse primer, a probe is used, and the nucleotide sequence of the probe is shown in SEQ ID No. 4;

[0015] When the primer combination described above is used, and the primer combination includes three forward primers and four reverse primers, four probes are used, and the nucleotide sequences of the probes are shown as SEQ ID No.4, SEQ ID No.10, SEQ ID No.11, and SEQ ID No.12.

[0016] When four probes are used, the nucleotide sequence of the probe shown in SEQ ID No.4, the fluorescent group labeled at the 5' end is VIC, and the quenching group labeled at the 3' end is BHQ1; the nucleotide sequence of the probe shown in SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12, the fluorescent group labeled at the 5' end is FAM, and the quenching group labeled at the 3' end is BHQ1.

[0017] The invention provides a kit for identifying Lycoris radiata mealybugs, comprising the primer combination and the probe.

[0018] The present invention provides a method for identifying mealybugs of Lycoris radiata, wherein the method for identifying mealybugs of Lycoris radiata is method (A) or method (B):

[0019] The method (A) comprises the following steps:

[0020] (a) extracting DNA from a sample to be tested as an amplification template;

[0021] (b) using fluorescent quantitative PCR or digital PCR for detection, the primer combination used for detection includes a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID No.2, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.3; the detection uses a probe, and the nucleotide sequence of the probe is shown in SEQ ID No.4;

[0022] (c) If an obvious fluorescent signal can be amplified, it means that the sample to be tested contains Lycoris radiata powdery mildew;

[0023] The method (B) comprises the following steps:

[0024] (I) extracting DNA from a sample to be tested as an amplification template;

[0025] (II) using a multiplex fluorescence quantitative PCR method for detection, the primer combination used for detection includes three forward primers and four reverse primers, the nucleotide sequences of the forward primers are shown in SEQ ID No.2, SEQ ID No.5 and SEQ ID No.6, and the nucleotide sequences of the reverse primers are shown in SEQ ID No.3, SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9; four probes are used for detection, and the nucleotide sequences of the probes are shown in SEQ ID No.4, SEQ ID No.10, SEQ ID No.11, and SEQ ID No.12;

[0026] (III) If both fluorescence channels can amplify signals, it means that the sample to be tested contains Lycoris radiata powdery mildew.

[0027] The fluorescent quantitative PCR reaction system is: II Probe qPCR SuperMix 10 μL, 5 pmol of each primer in the primer combination, 5 pmol of probe, 1 μL of template, and make up to 20 μL with sterile nuclease-free water; amplification reaction conditions are 94°C pre-denaturation for 30 s; 94°C denaturation for 5 s, 63.3°C annealing for 30 s, 72°C extension for 30 s, for a total of 40 cycles.

[0028] The digital PCR reaction system is: 10 μL of ddPCR Supermix for Probes, 18 pmol of each primer in the primer combination, 5 pmol of probe, 1 μL of template, and sterile nuclease-free water to 20 μL; the amplification reaction conditions are 95°C pre-denaturation for 10 min; 94°C denaturation for 30 s, 60°C annealing & extension for 60 s, for a total of 45 cycles; and maintaining 4°C after the reaction is completed.

[0029] The multiplex fluorescence quantitative PCR reaction system is: II Probe qPCR SuperMix 10 μL, 5 pmol of each primer in the primer combination, 5 pmol of the probe with a nucleotide sequence as shown in SEQ ID No.4, 2 pmol of each probe with a nucleotide sequence as shown in SEQ ID No.10 to 12, 1 μL of template, supplemented to 20 μL with sterile nuclease-free water; amplification reaction conditions are 94°C pre-denaturation for 30 s; 94°C denaturation for 5 s, 63.3°C annealing for 30 s, 72°C extension for 30 s, for a total of 40 cycles.

[0030] The invention is based on the high-throughput sequencing results of Lycoris mealybugs, screens the multi-copy specific target sequences on its genome, establishes a multi-channel fluorescence quantitative PCR detection method, and realizes the rapid identification of Lycoris mealybugs. The detection method of the invention has strong practicability, high accuracy, strong specificity, simple and rapid operation, and can meet the needs of relevant departments for inspection and quarantine or pest monitoring / detection.

[0031] The beneficial effects of the present invention are:

[0032] (1) The present invention provides a target or target combination for identifying Phenacoccus lycoris, wherein the target or target combination is derived from Phenacoccus lycoris;

[0033] (2) The present invention provides a method for identifying Lycoris radiata mealybugs, which is identified by multi-channel fluorescence quantitative PCR, which can improve the accuracy of the detection results and shorten the detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a graph showing the specific detection results of fluorescent quantitative PCR of a pair of primers and probes for Lycoris radiata mealybugs; among them, a is Lycoris radiata mealybugs, and b~1 are Bemisia tabaci, brown planthopper, pumpkin fruit fly, gypsy moth, citrus fruit fly, three-spotted stick-bellied fruit fly, striped fruit fly, hibiscus mealybugs, cassava mealybugs, new pineapple gray mealybugs, and citrus arm-striped mealybugs.

[0035] Figure 2 This is a graph showing the results of fluorescent quantitative PCR detection of genomic DNA of Phenacoccus lycoris using a pair of primer and probe combinations at different concentrations; among them, the concentrations represented by a to h are 57.07 ng / μl, 14.27 ng / μl, 3.57 ng / μl, 0.89 ng / μl, 0.23 ng / μl, 0.056 ng / μl, 0.014 ng / μl, and 0.0035 ng / μl, respectively.

[0036] Figure 3 This is the digital PCR detection result of Lycoris radiata powderycoides using a pair of primers and probe combinations.

[0037] Figure 4 The figure shows the specific detection results of fluorescent quantitative PCR of Phenacoccus lycoris using two pairs of primer and probe combinations; among them, a is Phenacoccus lycoris, and b~1 are Bemisia tabaci, Nilaparvata lugens, Cake fruit fly, Gypsy moth, Bactrocera dorsalis, Bactrocera trispinata, Bactrocera fasciatus, Phenacoccus hibiscus, Phenacoccus cassava, Phenacoccus neopineapple, and Phenacoccus citrus.

[0038] Figure 5Figure 1 shows the specific detection results of fluorescent quantitative PCR of Phenacoccus lycoris using primer and probe combination 1 and 2; A: VIC channel; B: FAM channel; a is Phenacoccus lycoris, b~1 are Bemisia tabaci, Nilaparvata lugens, Cake fruit fly, Gypsy moth, Bactrocera dorsalis, Bactrocera trispinata, Bactrocera fasciatus, Phenacoccus hibiscus, Phenacoccus cassava, Phenacoccus neopineapple, and Phenacoccus citrus. DETAILED DESCRIPTION

[0039] Example 1

[0040] High-throughput sequencing of the Phenacoccus lactis genome and design of fluorescent quantitative PCR primers and probes.

[0041] (1) Obtaining materials

[0042] Sterile nuclease-free water; upstream and downstream primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; blood & tissue DNA extraction kits were purchased from Qiagen Biotechnology (Shenzhen) Co., Ltd. Samples of Phenacoccus lycoris and Phenacoccus hibiscus were preserved by Zhejiang Institute of Inspection and Quarantine Science and Technology.

[0043] (2) DNA extraction of Phenacoccus spp.

[0044] The template DNA of Phenacoccus spp. was obtained using a blood & tissue DNA extraction kit (QIAGEN, Cat. No. 69504). The specific extraction steps are as follows:

[0045] 1) Place the Phenacoccus spp. sample in a 1.5 mL centrifuge tube, add 180 μL of Buffer ATL, and grind thoroughly with a grinding rod; then add 20 μL of proteinase K and vortex to mix;

[0046] 2) Add 200 μL of Buffer AL to a 1.5 mL centrifuge tube, vortex to mix, and place in a 56°C water bath for 10 min until the liquid becomes clear;

[0047] 3) Add 200 μL of ethanol (96%-100%, v / v) to a 1.5 mL centrifuge tube and vortex to mix;

[0048] 4) Transfer the solution to a DNeasy Mini column, place a 2 mL collection tube on the column, centrifuge at 8000 rpm for 1 min, and discard the waste liquid;

[0049] 5) Add 500 μL Buffer AW1 to the DNeasy Mini column, centrifuge at 8000 rpm for 1 min, and discard the waste liquid;

[0050] 6) Add 500 μL Buffer AW2 to the DNeasy Mini column, centrifuge at 14000 rpm for 3 min, and discard the waste liquid;

[0051] 7) Discard the collection tube and place the DNeasy Mini column into a new 1.5 mL centrifuge tube;

[0052] 8) Add 200 μL Buffer AE to the column for elution, let stand at 15-25°C for 1 min, and centrifuge at 8000 rpm for 1 min;

[0053] 9) Repeat step 8;

[0054] 10) After sufficient elution, store at -20°C for future use.

[0055] (3) High-throughput sequencing and sequence analysis

[0056] After random shearing, linker ligation, PCR amplification and other steps, sufficient DNA was obtained from the genomic DNA sample of Phenacoccus spp. for 2×150bp sequencing on the second-generation high-throughput sequencing platform Illumina Hiseq. The total sequencing volume was about 40 million reads, or 6 gillion bps.

[0057] bbmap, ABySS, GapCloser and other software were used to perform quality control, sequence splicing and result correction on sequencing results.

[0058] (4) Design of primers and probes for fluorescence quantitative PCR

[0059] The high-copy fragments were screened in the splicing results as candidate targets for identification of Phenacoccus lycoris, and the target sequence obtained by screening was shown in SEQ ID No. 1, which had no homology with any known sequence in the database. The target (target 1) as described in SEQ ID No. 1 was then compared with other Phenacoccus lycoris sequencing results, and fluorescent quantitative PCR primers and probes were designed at sites where there were significant differences in the sequences. The sequences of the primers and probes are shown in Table 1.

[0060] Table 1

[0061]

[0062] Example 2

[0063] Fluorescence quantitative PCR detection-primer and probe combination 1.

[0064] (1) Obtaining materials

[0065] Sterile nuclease-free water and PerfectStart II Probe qPCR SuperMix were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; upstream and downstream primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; blood & tissue DNA extraction kits were purchased from Qiagen Biotechnology (Shenzhen) Co., Ltd. Samples of Phenacoccus lycoris and Phenacoccus hibiscus were preserved by Zhejiang Institute of Inspection and Quarantine Science and Technology.

[0066] (2) DNA extraction

[0067] The DNA extraction procedure was the same as described in Example 1.

[0068] (3) Fluorescence quantitative PCR detection

[0069] The nucleotide sequences of the forward primer, reverse primer and probe used in the detection are shown as SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0070] Take out the frozen template DNA, primers, probes and other reagents in advance, restore them to room temperature (15-25°C), vortex mix and briefly centrifuge for use; prepare the fluorescence quantitative PCR system as shown in Table 2, and mix thoroughly after preparation; place the reaction tube in the detection well of the fluorescence quantitative amplification instrument; set the name of each reaction tube and other related labels; select the VIC channel for fluorescence signal detection; the reaction program is set as shown in Table 2.

[0071] Table 2

[0072] Reagents Usage Final concentration PerfectStart II Probe qPCR Super Mix 10μL 1x Forward primer 1 (10 μM) 0.5μL 0.25μM Reverse primer 1 (10 μM) 0.5μL 0.25μM Probe 1 (10 μM) 0.5μL 0.25μM DNA template 1μL Nuclease-Free Water Make up the system to 20 μL

[0073] Table 3

[0074]

[0075] (4) Effect of annealing temperature on detection results

[0076] The same concentration of Phenacoccus lycoris genomic DNA was tested at different annealing temperatures (55℃~65℃). The test results in Table 4 show that in the range of 55℃~63.3℃, ​​the test results are not greatly affected by the annealing temperature as the annealing temperature increases. Since nonspecific amplification decreases with the increase of annealing temperature, 63.3℃ is more recommended as the annealing temperature for fluorescent quantitative PCR. When multiple primers are tested, the annealing temperature can also be set according to the primers of other groups.

[0077] Table 4

[0078] Annealing temperature Cq value 65.0℃ 30.01 64.5℃ 27.70 63.3℃ 25.91 61.4℃ 25.28 59.0℃ 25.24 57.0℃ 25.12 55.7℃ 25.06 55.0℃ 25.32

[0079] (5) Primer specificity detection

[0080] The specificity of the primers was tested using genomic DNA of 11 insect species including Phenacoccus hibiscus and Phenacoccus cassava as well as Phenacococcus lycoris radiata as templates ( Figure 1 ). The reaction system and procedure were optimized according to the results of step (4), and the annealing temperature was 63.3°C. The results showed that the primer had obvious signals only in the Lycoris radiata mealybug group, and no signals in the other groups, which indicated that the primer and the detection method had good specificity. The Cq values ​​of the detection results are shown in Table 5.

[0081] Table 5

[0082] variety Cq value Bemisia tabaci N / A Brown Planthopper N / A Squash fruit fly N / A Gypsy moth N / A Bactrocera dorsalis N / A Bactrocera trispinata N / A Bactrocera fasciatus N / A Lycoris radiata mealybug 21.04 Phenacoccus hibiscus N / A Phenacoccus cassavaensis N / A New pineapple mealybug N / A Phenacoccus citri N / A

[0083] (6) Amplification efficiency and detection limit

[0084] The genomic DNA of Phenacoccus lycoris was diluted and tested in a gradient of 1:4. Figure 2 The logarithm of the diluted concentration was taken and linear regression analysis was performed with the Cq value. The regression curve was calculated as y = -1.0654 × log2X + 28.294, R 2 =0.992, where y is the Cq value of the test and X is the concentration of the genomic DNA of Phenacoccus lycoris (ng / μL). The amplification efficiency of primer and probe combination 1 was calculated to be 91.67%. When the sample concentration was 3.48×10 - When the concentration was 3 ng / μL, there was only a weak amplification signal and the reproducibility between different batches was poor. Therefore, the detection limit of this method was considered to be 3.48×10 -3 ng / μL.

[0085] The low amplification efficiency of this method is caused by the higher annealing temperature. Considering that increasing the annealing temperature can reduce the probability of false positives, it is believed that the slightly lower amplification efficiency is acceptable.

[0086] Example 3

[0087] Digital PCR Assay - Primer and Probe Combination 1.

[0088] (1) Obtaining materials

[0089] Sterile nuclease-free water and ddPCR Supermix for Probes (without dUTP) were purchased from Bio-Rad Biotech Co., Ltd.; upstream and downstream primer combinations and probe combinations were synthesized by Nanjing GenScript Biotech Co., Ltd.; blood & tissue DNA extraction kits were purchased from Qiagen Biotech (Shenzhen) Co., Ltd. Samples of Phenacoccus lycoris and Phenacoccus hibiscus were preserved by Zhejiang Institute of Inspection and Quarantine Science and Technology.

[0090] (2) DNA extraction

[0091] The DNA extraction procedure was the same as described in Example 1.

[0092] (3) Digital PCR detection

[0093] The nucleotide sequences of the forward primer, reverse primer and probe used in the detection are shown as SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0094] Take out the frozen template DNA, primers, probes and other reagents in advance, restore them to room temperature (15-25°C), vortex mix and briefly centrifuge for use; prepare the digital PCR system as shown in Table 6, and mix thoroughly after preparation;

[0095] Table 6

[0096]

[0097]

[0098] The prepared 20 μL reaction system was added to the droplet reaction card using a pipette; 70 μL of droplet generation oil was added to the droplet reaction card, the card rubber pad was covered, and the droplet generation card was placed in the droplet generator to generate droplets. After the droplets were generated, they were transferred to a 96-well PCR plate, the membrane was heat-sealed with a PX1 PCR heat sealer, and then placed on a digital PCR instrument for amplification. The reaction procedure is shown in Table 7. After the amplification reaction was completed, the 96-well PCR plate was placed in the QX 200 droplet analyzer, and the droplet reading, image processing and result analysis were performed using Quantasoft software. The detection channel was VIC.

[0099] Table 7

[0100]

[0101] (4) Analysis of the copy number of the target sequence in Phenacoccus lycoris

[0102] The DNA samples of Lycoris radiata powdery mildew were appropriately diluted and used for digital PCR detection. The results are as follows Figure 3 According to calculations, the number of copies of the target with the nucleotide sequence shown in SEQ ID No. 1 on a single Lycoris radiata Phenacoccus lycopersicus genome is about 5.

[0103] This method showed no obvious amplification signals in 11 insect species, including Bemisia tabaci, brown planthopper, pumpkin fruit fly, gypsy moth, citrus fruit fly, three-spotted fruit fly, striped fruit fly, hibiscus mealybug, cassava mealybug, new pineapple gray mealybug, and citrus arm-striped mealybug.

[0104] Example 4

[0105] Fluorescence quantitative PCR primer and probe design and experiment-primer and probe combination 2.

[0106] (1) Obtaining materials

[0107] Sterile nuclease-free water and PerfectStart II Probe qPCR SuperMix were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; upstream and downstream primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; blood & tissue DNA extraction kits were purchased from Qiagen Biotechnology (Shenzhen) Co., Ltd. Samples of Phenacoccus lycoris and Phenacoccus hibiscus were preserved by Zhejiang Institute of Inspection and Quarantine Science and Technology.

[0108] (2) Sequence analysis and primer and probe design

[0109] The mitochondrial COI gene of Phenacoccus lycoris was used as the detection target, and all COI gene sequences related to mealybugs were retrieved in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). All COI sequences of Phenacoccus lycoris were compared with those of other mealybugs. Considering the polymorphism within the species of Phenacoccus lycoris and the differences between Phenacoccus lycoris and other species, the primer combinations were designed as shown in Table 8. Using primer and probe combinations for amplification can effectively avoid false negatives caused by polymorphism within species.

[0110] Table 8

[0111]

[0112] (3) DNA extraction

[0113] The DNA extraction procedure was the same as described in Example 1.

[0114] (4) Fluorescence quantitative PCR detection

[0115] The nucleotide sequences of the forward primer combination used for detection are shown in SEQ ID No.5 and SEQ ID No.6, the nucleotide sequences of the reverse primer combination are shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, and the nucleotide sequences of the probe combination are shown in SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12.

[0116] The detection target (target 2) sequence of the above primers and probes is shown in SEQ ID No.13.

[0117] Take out the frozen template DNA, primers, probes and other reagents in advance, restore them to room temperature (15-25°C), vortex mix and briefly centrifuge for use; prepare the fluorescence quantitative PCR system as shown in Table 9, and mix it thoroughly after preparation; place the reaction tube in the detection well of the fluorescence quantitative amplification instrument; set the name of each reaction tube and other related labels; select the FAM channel for fluorescence signal detection; set the reaction program as shown in Table 3.

[0118] Table 9

[0119]

[0120]

[0121] (5) Effect of annealing temperature on detection results

[0122] The same concentration of Phenacoccus lycoris genomic DNA was tested at different annealing temperatures (55°C to 65°C). The test results in Table 10 show that in the range of 55.0°C to 65.0°C, the test results are not greatly affected by the annealing temperature. Since nonspecific amplification decreases with increasing annealing temperature, 65.0°C is recommended as the annealing temperature for fluorescent quantitative PCR. When multiple primers are tested, the annealing temperature can also be set according to the primers of other groups.

[0123] Table 10

[0124] Annealing temperature Cq value 65.0℃ 24.59 64.5℃ 24.13 63.3℃ 23.97 61.4℃ 23.91 59.0℃ 23.80 57.0℃ 23.97 55.7℃ 24.07 55.0℃ 24.16

[0125] (6) Primer specificity detection

[0126] The specificity of the primers was tested using genomic DNA of 11 insect species including Phenacoccus hibiscus and Phenacoccus cassava as well as Phenacococcus lycoris radiata as templates ( Figure 4 ). The reaction system and procedure were optimized according to the results of step (5), and the annealing temperature was 65.0°C. The results showed that the primer had obvious signals only in the Lycoris radiata mealybug group, and no signals were found in the other groups, which indicated that the primer and the detection method had good specificity. The Cq values ​​of the detection results are shown in Table 11.

[0127] Table 11

[0128] variety Cq value Bemisia tabaci N / A Brown Planthopper N / A Squash fruit fly N / A Gypsy moth N / A Bactrocera dorsalis N / A Bactrocera trispinata N / A Bactrocera fasciatus N / A Lycoris radiata mealybug 21.31 Phenacoccus hibiscus N / A Phenacoccus cassavaensis N / A New pineapple mealybug N / A Phenacoccus citri N / A

[0129] Example 5

[0130] Multiplex fluorescence quantitative PCR

[0131] (1) Sterile nuclease-free water and PerfectStart II Probe qPCR SuperMix were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; upstream and downstream primer combinations and probe combinations were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; blood and tissue DNA extraction kits were purchased from Qiagen Biotechnology (Shenzhen) Co., Ltd. Samples of Phenacoccus lycoris and Phenacoccus hibiscus were preserved by Zhejiang Institute of Inspection and Quarantine Science and Technology.

[0132] (2) DNA extraction

[0133] The DNA extraction procedure was the same as described in Example 1.

[0134] (3) Fluorescence quantitative PCR detection

[0135] The nucleotide sequences of the forward primer, reverse primer and probe used for detecting target 1 are shown in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0136] The nucleotide sequences of the forward primer combination used for target 2 detection are shown in SEQ ID No.5 and SEQ ID No.6, the nucleotide sequences of the reverse primer combination are shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, and the nucleotide sequences of the probe combination are shown in SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12.

[0137] Primer and probe combination 1 can amplify well within the annealing temperature range of 55.0-63.3° C., and primer and probe combination 2 can amplify well within the annealing temperature range of 55.0-65.0° C. Considering both situations, 63.3° C. is selected as the annealing temperature.

[0138] Take out the frozen template DNA, primers, probes and other reagents in advance, restore them to room temperature (15-25°C), vortex mix and briefly centrifuge for use; prepare the fluorescence quantitative PCR system as shown in Table 12, and mix thoroughly after preparation; place the reaction tube in the detection well of the fluorescence quantitative amplification instrument; set the name of each reaction tube and other related labels; select VIC and FAM channels for fluorescence signal detection at the same time; set the reaction program as shown in Table 13.

[0139] Table 12

[0140]

[0141] Table 13

[0142]

[0143]

[0144] (4) Specificity detection

[0145] Table 14

[0146] variety VIC channel Cq value FAM channel Cq value Bemisia tabaci N / A N / A Brown Planthopper 38.16 N / A Squash fruit fly 36.42 N / A Gypsy moth N / A N / A Bactrocera dorsalis N / A N / A Bactrocera trispinata N / A N / A Bactrocera fasciatus N / A N / A Lycoris radiata mealybug 16.21 16.01 Phenacoccus hibiscus 37.64 N / A Phenacoccus cassavaensis N / A N / A New pineapple mealybug N / A N / A Phenacoccus citri N / A N / A

[0147] The specificity of the primers was tested using genomic DNA of 11 insect species including Phenacoccus hibiscus and Phenacoccus cassava as well as Phenacococcus lycoris radiata as templates ( Figure 5 ). Lycoris radiata mealybug has obvious signals in both VIC and FAM channels; the Cq values ​​of the detection results are shown in Table 14. When the sample concentration is high, weak models can be detected in the VIC channel of brown planthopper, pumpkin fruit fly, and hibiscus mealybug, but the Cq values ​​are all greater than 35, and the three have no obvious signals in the FAM channel. This shows that the multiplex fluorescence quantitative PCR detection method has good specificity.

[0148] Compared with single-channel fluorescence quantitative PCR, multiplex fluorescence quantitative PCR can effectively improve the accuracy of identification results. When both channels have obvious signals and the Cq value is less than 35, it can be confirmed that the sample is Phenacoccus lycoris.

Claims

1. A kit for identifying Lycoris radiata mealybugs, characterized in that: Including primer combinations and probes; The primer combination includes three forward primers and four reverse primers, the nucleotide sequence of the forward primer combination is shown as SEQ ID No.2, SEQ ID No.5 and SEQ ID No.6, the nucleotide sequence of the reverse primer combination is shown as SEQ ID No.3, SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, and the nucleotide sequence of the probe is shown as SEQ ID No.4, SEQ ID No.10, SEQ ID No.11 and SEQ ID No.

12.

2. A kit for identifying Phenacoccus lycoris as claimed in claim 1, characterized in that: When four probes are used, the nucleotide sequence of the probe shown in SEQ ID No.4, the fluorescent group labeled at the 5' end is VIC, and the quenching group labeled at the 3' end is BHQ1; the nucleotide sequence of the probe shown in SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12, the fluorescent group labeled at the 5' end is FAM, and the quenching group labeled at the 3' end is BHQ1.

3. A method for identifying Phenacoccus lycoris, characterized in that: The method for identifying the powdery mildew of Lycoris radiata comprises the following steps: (I) extracting DNA from the sample to be tested as an amplification template; (II) using a multiplex fluorescence quantitative PCR method for detection, the primer combination used for detection includes three forward primers and four reverse primers, the nucleotide sequences of the forward primers are shown in SEQ ID No.2, SEQ ID No.5 and SEQ ID No.6, and the nucleotide sequences of the reverse primers are shown in SEQ ID No.3, SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9; four probes are used for detection, and the nucleotide sequences of the probes are shown in SEQ ID No.4, SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12; Among them, for the probe whose nucleotide sequence is shown in SEQ ID No.4, the fluorescent group labeled at the 5' end is VIC, and the quenching group labeled at the 3' end is BHQ1; for the probe whose nucleotide sequence is shown in SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12, the fluorescent group labeled at the 5' end is FAM, and the quenching group labeled at the 3' end is BHQ1; (III) If both fluorescence channels can amplify signals, it means that the sample contains Lycoris radiata powdery mildew.

4. The method for identifying the powdery mildew of Lycoris radiata as claimed in claim 3, wherein: The multiplex fluorescence quantitative PCR reaction system is: IIProbe qPCR SuperMix 10 μL, 5 pmol of each primer in the primer combination, 5 pmol of the probe with a nucleotide sequence as shown in SEQ ID No.4, 2 pmol of each probe with a nucleotide sequence as shown in SEQ ID No.10 to 12, 1 μL of template, made up to 20 μL with sterile nuclease-free water; amplification reaction conditions were 94°C pre-denaturation for 30 s; 94°C denaturation for 5 s, 63.3°C annealing for 30 s, 72°C extension for 30 s, for a total of 40 cycles.

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