CN115786531B

CN115786531B - Method for identifying lycoris radiata mealy bugs and application

Info

Publication number: CN115786531B
Application number: CN202211099153.7A
Authority: CN
Inventors: 吴志毅; 张莉丽; 范凌; 季淑枫; 赵丽; 梁琨; 党志浩; 徐淼锋; 黄芳
Original assignee: Hangzhou Agricultural Technology Extension Center; Zhejiang Academy Of Science & Technology For Inspection & Quarantine
Current assignee: Hangzhou Agricultural Technology Extension Center; Zhejiang Academy Of Science & Technology For Inspection & Quarantine
Priority date: 2022-09-08
Filing date: 2022-09-08
Publication date: 2023-07-21
Anticipated expiration: 2042-09-08
Also published as: CN115786531A

Abstract

The invention discloses a method for identifying lycoris radiata mealy bugs and application thereof, and relates to the field of molecular biology. The target is a single-sequence target or a target combination consisting of two sequences, and the nucleotide sequence of the single-sequence target is shown as SEQ ID No. 1; the nucleotide sequences of the target combinations are shown as SEQ ID No.1 and SEQ ID No. 12. The invention provides a method for identifying lycoris radiata mealy scale, which can improve the accuracy of detection results and shorten the detection time by carrying out the identification through multichannel fluorescent quantitative PCR.

Description

Method for identifying lycoris radiata mealy bugs and application

Technical Field

The invention relates to the field of molecular biology, in particular to a method for identifying lycoris radiata mealy bugs and application thereof.

Background

Lycoris radiata (Phenacoccus solani) Ferris, belonging to the genus Hemiptera (Hemiptera), pinaceae (pseudococidae), pink mealy (Phenacoccus); female adults are elliptic, thick white wax powder is coated on the body surface, the color of the female adults is deep, obvious wax process at the body edge is provided, and the feet are deep red. The egg is oblong, yellow and transparent. The nymphs are elliptic, the body surface is coated with a layer of white thin wax powder, and after the thin wax powder layer is removed, the nymphs are yellow. The nymphs of first instar are compound eye balls and reddish brown; tooth-shaped protrusions are arranged on the body edges of the second-instar nymphs; the third-instar nymphs have obvious body edge protrusions, the tail petals protrude, and the color of feet is darker than that of the third-instar nymphs.

The lycoris radiata mealy is a small insect, is shaped like female and male, is egg-shaped, is white wax powder, has a body length of 2.3-2.7mm and a body width of 1.3-1.6mm. Parthenogenesis and ampholytic reproduction can be achieved. Female adults lay eggs in cotton-shaped oocysts near the reproductive holes, the hatched nymphs are active, and the female adults can eat the eggs within a short time after the eggs climb out of the oocysts. The lycoris radiata mealy scale has extremely strong reproductive capacity, the egg laying amount of adults is high, the generation of the population is overlapped, and the population can grow at extremely high speed. Feeding sites of nymphs of each age were not fixed: the second-instar nymphs mostly feed on stems, flower buds and leaf axils of parasitic plants; fixing the feeding place of the nymphs of three ages; adults colonize the plant stems and occasionally the leaf backs of the host plants. Lycoris radiata mealy is used for sucking juice into plant tissues by utilizing an elongated oral needle to puncture the plant tissues, so that cells and some nutrition-dredging tissues in the plant are destroyed, and the plant is weakened, the tissues are recessed and even wilted and dead. Lycoris glabra mealy also can secrete a large amount of honeydew to induce host plants to generate coal pollution, reduce photosynthesis of the plants, cause poor growth of the host plants, promote early leaf and fruit fall and reduce yield.

The lycoris glaucopia host plant has wide range, and the reported host plant comprises 31 families of orchidaceae, compositae, leguminosae, zingiberaceae, acanthaceae, new Zealand, araliaceae, amaryllidaceae, etc., wherein the families of Amaryllidaceae, compositae, amaryllidaceae, etc. The lycoris longipes can be spread along with host plants and related products thereof. In the overseas entry plants, lycoris radiata mealy bugs need to be inspected and quarantined. However, since the individual lycoris radiata is tiny and the surface is covered with a layer of wax powder, the specific characteristics of the body surface cannot be directly observed by a stereoscopic microscope.

The current international method for identifying lycoris radiata mealy scale mainly comprises the following steps: 1) Morphological feature identification; 2) PCR techniques, and the like. The morphological characteristic identification needs to be carried out on the whole insect body, the identification process is complex, and certain technical difficulty exists; the PCR technology carries out classification and identification on the mealybugs through DNA sequences, and the result is accurate, but the time required for sequencing is long. Therefore, in order to prevent the penetration of lycoris radiata mealy bugs, the invention has important social and economic significance for a rapid, high-sensitivity and high-specificity identification detection method of the mealy bugs. By utilizing the detection method, lycoris radiata mealy bugs can be effectively detected, and the detection rate is improved; the method is helpful for pertinently preventing the invasion and the diffusion of lycoris radiata, and protecting the agricultural production safety and the ecological safety of China.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a method for identifying lycoris radiata and application thereof.

The invention provides a target for molecular biological identification of lycoris radiata (Phenacoccus solani), wherein the target is a single-sequence target or a target combination consisting of two sequences, and the nucleotide sequence of the single-sequence target is shown as SEQ ID No. 1; the nucleotide sequences of the target combinations are shown as SEQ ID No.1 and SEQ ID No. 12.

The invention also provides application of the target in identifying lycoris radiata mealy bugs.

The invention also provides a primer combination for identifying lycoris radiata mealy bugs,

the primer combination comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID No.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 3;

alternatively, the primer combination comprises 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.

The invention also provides a probe for identifying lycoris radiata mealy bugs,

when the primer combination is used and comprises a forward primer and a reverse primer, a probe is used, and the nucleotide sequence of the probe is shown as SEQ ID No. 4;

when the primer combination described above is used and the primer combination includes three forward primers and four reverse primers, three probes are used, the nucleotide sequences of which are shown in SEQ ID No.4, SEQ ID No.10 and SEQ ID No. 11.

When three probes are used, the nucleotide sequence of the probe is shown as SEQ ID No.4, the fluorescent group marked at the 5 'end is FAM, and the quenching group marked at the 3' end is BHQ1; the nucleotide sequences of the probes are shown as SEQ ID No.10 and SEQ ID No.11, the fluorescent group marked at the 5 'end is ROX, and the quenching group marked at the 3' end is BHQ1.

The invention also provides a kit for identifying lycoris radiata, which comprises the primer combination and the probe.

The invention also provides a method for identifying lycoris radiata mealy bugs, which is a method (A) or a method (B):

the method (A) comprises the following steps:

(a) Extracting DNA of a sample to be detected as an amplification template;

(b) Detecting by using a fluorescent quantitative PCR or digital PCR method, wherein a primer combination used for detection comprises a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID No.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 3; the detection uses a probe, and the nucleotide sequence of the probe is shown as SEQ ID No. 4;

(c) If the sample can be amplified to obtain an obvious fluorescent signal, the sample to be detected contains lycoris radiata;

the method (B) comprises the steps of:

(I) Extracting DNA of a sample to be detected as an amplification template;

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

and (III) if the two fluorescent channels are amplified to obtain obvious signals, indicating that the sample to be detected contains lycoris mealy bugs.

The fluorescent quantitative PCR reaction system comprises the following components:II Probe qPCR SuperMix 10. Mu.L of each of 5pmol of the primers in the primer set, 5pmol of the probe, 1. Mu.L of the template, and 20. Mu.L of the template were filled with sterile nuclease-free water; the amplification reaction conditions were 94℃for 30s of pre-denaturation; denaturation at 94℃for 5s, annealing at 61.4℃for 30s, extension at 72℃for 30s for 40 cycles.

The digital PCR reaction system comprises: ddPCR Supermix for Probes 10. Mu.L of each of the primers in the primer set was 18pmol, 5pmol of probe, 1. Mu.L of template, and 20. Mu.L of sterile nuclease-free water; the amplification reaction condition is that the pre-denaturation is carried out for 10min at 95 ℃; denaturation at 94℃for 30s, annealing at 60℃for 60s and extension for 45 cycles; after the reaction was completed, the temperature was kept at 4 ℃.

The multiplex fluorescence quantitative PCR reaction system comprises:II Probe qPCR SuperMix 10. Mu.L of each primer in the primer combination, 5pmol of each probe with the nucleotide sequence shown as SEQ ID No.4, 3pmol of each probe with the nucleotide sequences shown as SEQ ID No.10 and SEQ ID No.11, and 1. Mu.L of template are complemented to 20. Mu.L by sterile nuclease-free water; the amplification reaction conditions were 94℃for 30s of pre-denaturation; denaturation at 94℃for 5s, annealing at 61.4℃for 30s, extension at 72℃for 30s for 40 cycles.

The invention screens the multi-copy specific target sequence on the genome based on the high-throughput sequencing result of lycoris radiata, establishes a multichannel fluorescent quantitative PCR detection method and realizes the rapid identification of lycoris radiata. The detection method has the advantages of strong practicability, high accuracy, strong specificity and simple and rapid operation, and can meet the requirements of related departments on inspection and quarantine or pest monitoring/detection.

The beneficial effects of the invention are as follows:

(1) The invention provides a target or a target combination for identifying lycoris radiata, wherein the target or the target combination is derived from lycoris radiata;

(2) The invention provides a method for identifying lycoris radiata mealy scale, which is characterized in that the accuracy of a detection result can be improved and the detection time can be shortened by carrying out the identification through multichannel fluorescent quantitative PCR.

Drawings

FIG. 1 is a diagram showing the result of fluorescent quantitative PCR specificity detection of primer and probe combinations 1 pair of lycoris mealy bugs; wherein a is lycoris mealybugs, and b-l are sequentially bemisia tabaci, brown planthoppers, pumpkin fruit flies, gypsy moths, citrus fruit flies, three-point rod fruit flies, bactrocera dorsalis, hibiscus rosa-sinensis mealybugs, cassava mealybugs, new pineapple mealybugs and citrus armyworms.

FIG. 2 is a diagram showing the result of fluorescent quantitative PCR detection of genomic DNA of lycoris radiata in different concentrations by using the primer and probe combination 1; the concentrations represented by a to h were 51.03 ng/. Mu.l, 12.76 ng/. Mu.l, 3.19 ng/. Mu.l, 0.79 ng/. Mu.l, 0.20 ng/. Mu.l, 0.050 ng/. Mu.l, 0.012 ng/. Mu.l, and 0.0031 ng/. Mu.l, in this order.

FIG. 3 is a graph showing the results of digital PCR detection of 1 pair of lycoris radiata by primer and probe combinations.

FIG. 4 is a diagram showing the result of fluorescence quantitative PCR specificity detection of 2 pairs of lycoris radiata by primer and probe combinations; wherein a is lycoris mealybugs, and b-l are sequentially bemisia tabaci, brown planthoppers, pumpkin fruit flies, gypsy moths, citrus fruit flies, three-point rod fruit flies, bactrocera dorsalis, hibiscus rosa-sinensis mealybugs, cassava mealybugs, new pineapple mealybugs and citrus armyworms.

FIG. 5 is a graph showing the results of fluorescent quantitative PCR specific detection of Amaryllidar mealy bugs using primer and probe combinations 1 and 2 in combination; a: FAM channels; b: a ROX channel; a is lycoris mealybugs, and b-l are sequentially bemisia tabaci, brown planthoppers, pumpkin fruit flies, gypsymoth, citrus fruit flies, three-point rod fruit flies, bactrocera dorsalis, hibiscus velutissima, cassava mealybugs, new pineapple mealybugs and citrus arm mealybugs.

Detailed Description

Example 1

High-throughput sequencing of the mealybugs genome and fluorescent quantitative PCR primer and probe design.

(1) Acquisition of material

Sterile nuclease-free water; the upstream and downstream primers and probes are synthesized by Nanjing Jinsri biotechnology limited company; blood & tissue DNA extraction kit was purchased from Kaemp bioengineering (Shenzhen) Inc. Samples of lycoris glaucopia and the like are preserved by Zhejiang province inspection and quarantine scientific and technical institute.

(2) Extraction of Ericerus pela DNA

The template DNA of mealybugs is obtained by using a blood & tissue DNA extraction kit (QIAGEN, cat.No.69504), and the specific extraction steps are as follows:

1) Placing a mealy bugs sample into a 1.5mL centrifuge tube, adding 180 mu L Buffer ATL, and fully grinding by using a grinding rod; then adding 20 mu L of proteinase K, and mixing by vortex;

2) 200 mu L Buffer AL is added into a 1.5mL centrifuge tube, vortex mixing is carried out, water bath is carried out for 10min at 56 ℃ until liquid is clear;

3) 200 mu L of ethanol (96% -100%, v/v) is added into a 1.5mL centrifuge tube, and vortex mixing is carried out;

4) Transferring the solution to DNeasy Mini column, sleeving the column with 2mL collecting tube, centrifuging at 8000rpm for 1min, and discarding the waste liquid;

5) Add 500. Mu.L Buffer AW to DNeasy Mini column ₁ Centrifuging at 8000rpm for 1min, and discarding the waste liquid;

6) Add 500. Mu.L Buffer AW to DNeasy Mini column ₂ Centrifuging at 14000rpm for 3min, and discarding the waste liquid;

7) Discarding the collection tube, placing the DNeasy Mini column into a new 1.5mL centrifuge tube;

8) Adding 200 mu L Buffer AE into the column for eluting, standing for 1min at 15-25 ℃ and centrifuging for 1min at 8000 rpm;

9) Repeating the step 8;

10 After sufficient elution, the mixture is preserved at the temperature of minus 20 ℃ for standby.

(3) High throughput sequencing and sequence analysis

The genomic DNA sample of the mealybugs is subjected to random breaking, joint connection, PCR amplification and other steps to obtain enough DNA for a second-generation high-throughput sequencing platform Illumina Hiseq to carry out 2×150bp sequencing. The total amount of sequencing was about 40million reads, i.e., 6gillion bps.

And using bbmap, ABySS, gapCloser and other software to control the quality of the sequencing result, splice the sequence and correct the result.

(4) Fluorescent quantitative PCR primer and probe design

And (3) screening a high-copy fragment in the splicing result to serve as a candidate target for identification of lycoris radiata, wherein the screened target sequence is shown as SEQ ID No.1 (target 1), and the sequence has no homology with known sequences in a database. Then comparing the target sequence shown as SEQ ID No.1 with other mealybugs sequencing results, and designing fluorescent quantitative PCR primers and probes at the sites with obvious difference in sequence. The sequences of the primers and probes are shown in Table 1.

TABLE 1

Example 2

Fluorescent quantitative PCR detection-primer and probe combination 1.

(1) Acquisition of material

Sterile nuclease-free water, perfectStart II Probe qPCR SuperMix, available from beijing all gold biotechnology limited; the upstream and downstream primers and probes are synthesized by Nanjing Jinsri biotechnology limited company; blood & tissue DNA extraction kit was purchased from Kaemp bioengineering (Shenzhen) Inc. Samples of lycoris glaucopia and the like are preserved by Zhejiang province inspection and quarantine scientific and technical institute.

(2) DNA extraction

The DNA extraction procedure was the same as described in example 1.

(3) Fluorescent quantitative PCR detection

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

Taking out the frozen and preserved template DNA, primer, probe and other reagents in advance, and after the temperature is restored to room temperature (15-25 ℃), carrying out vortex mixing and short-term centrifugation for later use; preparing a fluorescent quantitative PCR system as shown in Table 2, and fully and uniformly mixing after the preparation is completed; placing the reaction tube in a detection hole of a fluorescent quantitative amplification instrument; setting related marks such as names of the reaction tubes; selecting FAM channel to detect fluorescence signal; the settings of the reaction procedure are shown in Table 2.

TABLE 2

Reagent(s)	Usage amount	Final concentration
			PerfectStart II Probe qPCR Super Mix	10μL	1x
Forward primer 1 (10. Mu.M)	0.5μL	0.25μM
			Reverse primer 1 (10. Mu.M)	0.5μL	0.25μM
Probe 1 (10 mu M)	0.5μL	0.25μM
			DNA template	1μL
Non-enzymatic Water (Nuclear-Free Water)	Make up the system to 20. Mu.L

TABLE 3 Table 3

(4) Influence of annealing temperature on detection results

The genomic DNA of lycoris radiata powder with the same concentration is detected aiming at different annealing temperatures (55-65 ℃). The results of the detection in Table 4 show that the Cq value of the detection result is the lowest in the range of 55℃to 61.4℃and the detection result is not greatly affected by the annealing temperature as the annealing temperature increases, and that the non-specific amplification is decreased as the annealing temperature increases, so that the annealing temperature of the fluorescent quantitative PCR is more preferably 61.4 ℃. When multiple detection is performed on a plurality of primers, the annealing temperature may be set according to the other sets of primers.

TABLE 4 Table 4

(5) Primer-specific detection

The specificity of the primers was detected using genomic DNA of 11 insects such as Hibiscus sabdariffa, gecko manihot, etc. as templates, respectively (fig. 1). The system and procedure of the reaction were optimized according to the result of step (4), and the annealing temperature was 61.4 ℃. The results show that the primer only has obvious signals in the lycoris radiata group and no signals exist in the other groups, thereby indicating that the primer and the detection method have good specificity. The measurement result Cq values are shown in table 5.

TABLE 5

Variety of species	Cq value
		Bemisia tabaci (Bemisia tabaci)	N/A
Brown planthopper	N/A
		Pumpkin fly	N/A
Lymantria dispar	N/A
		Bactrocera dorsalis (L.) Kuntze	N/A
Three-point roller drosophila melanogaster	N/A
		Bactrocera dorsalis with stripes	N/A
Lycoris radiata mealybugs	20.71
		Hibiscus-sinensis gecko	N/A
Cassava mealybugs	N/A
		New pineapple gray mealybugs	N/A
Orange arm-print mealybugs	N/A

(6) Amplification efficiency and detection limit

The genomic DNA of Amaryllidar was diluted and examined according to a 1:4 ratio gradient (FIG. 2). The diluted concentration is subjected to linear regression analysis with Cq value, and a regression curve is calculated as y= -0.9821 ×log ₂ X+28.005，R ² =0.9969, where y is the Cq value detected and X is the concentration of lycoris mealy bugs genomic DNA (ng/μl). The amplification efficiency of primer and probe combination 1 was calculated to be 102.54%. When the sample concentration was 3.11X10 ^-3 At ng/. Mu.L, there is only a weak amplification signal and the reproducibility between batches is poor, so this method is considered to have a detection limit of 3.11X10 ^-3 ng/μL。

Example 3

Digital PCR detection-primer and probe combination 1.

(1) Acquisition of material

Sterile nuclease-free water, ddPCR Supermix for Probes (dUTP-free) was purchased from buret bioengineering limited; the upstream and downstream primer combinations and the probe combinations are synthesized by Nanjing Jinsri biotechnology limited company; blood & tissue DNA extraction kit was purchased from Kaemp bioengineering (Shenzhen) Inc. Samples of lycoris glaucopia and the like are preserved by Zhejiang province inspection and quarantine scientific and technical institute.

(2) DNA extraction

The DNA extraction procedure was the same as described in example 1.

(3) Digital PCR detection

Taking out the frozen and preserved template DNA, primer, probe and other reagents in advance, and after the temperature is restored to room temperature (15-25 ℃), carrying out vortex mixing and short-term centrifugation for later use; preparing a digital PCR system as shown in Table 6, and fully and uniformly mixing after the preparation is completed;

TABLE 6

Adding the prepared 20 mu L reaction system into a microdroplet reaction card by using a pipettor; 70. Mu.L of droplet generation oil was added to the droplet reaction card, the pad of the generating card was covered, and the droplet generation card was placed in the droplet generator to generate droplets. After droplet generation, it was transferred to a 96-well PCR plate, membrane heat sealed with a PX1 PCR heat sealer, and amplified on a digital PCR machine, with the reaction procedure as shown in table 7. After the amplification reaction is completed, the 96-well PCR plate is placed in a QX 200 droplet analyzer, and droplet reading, image processing and result analysis are performed using quanthaoft software. The detection channel is FAM.

TABLE 7

(4) Copy number analysis of target sequences in lycoris mealy bugs

The lycoris glabra DNA sample is properly diluted and used for digital PCR detection, and the result is shown in figure 3. According to calculation, the copy number of the target with the nucleotide sequence shown as SEQ ID No.1 on a single lycoris mealy scale genome is about 3.

The method has no obvious amplified signals in 11 insects of Bemisia tabaci, brown planthoppers, pumpkin fruit flies, gypsymoth, bactrocera dorsalis, three-point rod belly fruit flies, bactrocera dorsalis, hibiscus sabdariffa, pimenta sinensis, and Lepidoptera citri.

Example 4

Fluorescent quantitative PCR primer and probe design and assay-primer and probe combination 2.

(1) Acquisition of material

(2) Sequence analysis and primer and probe design

Mitochondrial COI genes of lycoris mealy bugs are used as detection targets, and all mealy bugs related COI gene sequences are searched in NCBI database (https:// www.ncbi.nlm.nih.gov /). All the COI sequences of lycoris mealy bugs and the COI sequences of other mealy bugs were compared with each other, and primer combinations were designed as shown in table 8, taking into account the polymorphism in lycoris mealy bugs species and the differences between lycoris mealy bugs and other species. The primer and probe combination is used for amplification, so that the polymorphism in the lycoris radiata can be covered, and the occurrence of false negative is reduced.

TABLE 8

(3) DNA extraction

The DNA extraction procedure was the same as described in example 1.

(4) Fluorescent quantitative PCR detection

The nucleotide sequence of the forward primer combination used for detection is shown as SEQ ID No.5 and SEQ ID No.6, the nucleotide sequence of the reverse primer combination is shown as SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, and the nucleotide sequence of the probe combination is shown as SEQ ID No.10 and SEQ ID No. 11.

The sequence of the detection target (target 2) of the primer and the probe is shown as SEQ ID No. 12.

Taking out the frozen and preserved template DNA, primer, probe and other reagents in advance, and after the temperature is restored to room temperature (15-25 ℃), carrying out vortex mixing and short-term centrifugation for later use; preparing a fluorescent quantitative PCR system as shown in Table 9, and fully and uniformly mixing after the preparation is completed; placing the reaction tube in a detection hole of a fluorescent quantitative amplification instrument; setting related marks such as names of the reaction tubes; selecting a ROX channel to detect a fluorescent signal; the settings of the reaction procedure are shown in Table 3.

TABLE 9

(5) Influence of annealing temperature on detection results

The genomic DNA of lycoris radiata powder with the same concentration is detected aiming at different annealing temperatures (55-65 ℃). The test results in Table 10 show that the test results are not greatly affected by the annealing temperature in the range of 55.0℃to 61.4℃as the annealing temperature increases, and that the non-specific amplification is decreased with the increase of the annealing temperature, so that the annealing temperature of the fluorescent quantitative PCR is more preferably 61.4 ℃. When multiple detection is performed on a plurality of primers, the annealing temperature may be set according to the other sets of primers.

Table 10

Annealing temperature	Cq value
		65.0℃	30.27
64.5℃	28.63
		63.3℃	26.00
61.4℃	25.28
		59.0℃	25.06
57.0℃	25.08
		55.7℃	25.15
55.0℃	25.19

(6) Primer-specific detection

The specificity of the primers was detected using genomic DNA of 11 insects such as Hibiscus sabdariffa, gecko manihot, etc., as templates, respectively (fig. 4). The system and procedure of the reaction were optimized according to the result of step (5), and the annealing temperature was 61.4 ℃. The results show that the primer only has obvious signals in the lycoris radiata group and no signals exist in the other groups, thereby indicating that the primer and the detection method have good specificity. The measurement result Cq values are shown in table 11.

TABLE 11

Example 5

Multiplex fluorescent quantitative PCR

(1) Sterile nuclease-free water, perfectStart II Probe qPCR SuperMix, available from beijing all gold biotechnology limited; the upstream and downstream primer combinations and the probe combinations are synthesized by Nanjing Jinsri biotechnology limited company; blood & tissue DNA extraction kit was purchased from Kaemp bioengineering (Shenzhen) Inc. Samples of lycoris glaucopia and the like are preserved by Zhejiang province inspection and quarantine scientific and technical institute.

(2) DNA extraction

The DNA extraction procedure was the same as described in example 1.

(3) Fluorescent quantitative PCR detection

The nucleotide sequences of the forward primer, the reverse primer and the probe used for target 1 detection are respectively shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.

The nucleotide sequence of the forward primer combination used for target 2 detection is shown as SEQ ID No.5 and SEQ ID No.6, the nucleotide sequence of the reverse primer combination is shown as SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, and the nucleotide sequence of the probe combination is shown as SEQ ID No.10 and SEQ ID No. 11.

The primer and probe combination 1 can be amplified well at the annealing temperature of 55.0-61.4 ℃, and the primer and probe combination 2 can be amplified well at the annealing temperature of 55.0-61.4 ℃. And (3) combining the two conditions, and selecting 61.4 ℃ as the annealing temperature.

Taking out the frozen and preserved template DNA, primer, probe and other reagents in advance, and after the temperature is restored to room temperature (15-25 ℃), carrying out vortex mixing and short-term centrifugation for later use; preparing a fluorescent quantitative PCR system as shown in Table 12, and fully and uniformly mixing after the preparation is completed; placing the reaction tube in a detection hole of a fluorescent quantitative amplification instrument; setting related marks such as names of the reaction tubes; simultaneously selecting FAM and ROX channels to detect fluorescence signals; the settings of the reaction procedure are shown in Table 13.

Table 12

TABLE 13

(4) Specific detection

TABLE 14

/>

The specificity of the primers was detected using genomic DNA of 11 insects such as Hibiscus sabdariffa, gecko manihot, etc., as templates, respectively (fig. 5). Lycoris glabra mealy bugs have obvious signals in FAM and ROX channels; the Cq value of the detection result is shown in Table 14, when the concentration of the sample is high, the FAM channel of the cassava mealybugs and the ROX channel of the Hibiscus manihot mealybugs can detect weak models, but the Cq value is larger than 35, and signals are not detected in the FAM channel and the ROX channel at the same time. The specificity of the multiplex fluorescence quantitative PCR detection method is good.

Compared with single-channel fluorescent quantitative PCR, the multiplex fluorescent quantitative PCR can effectively improve the accuracy of the identification result, and when the two channels have obvious signals and the Cq value is smaller than 35, the sample can be confirmed to be lycoris mealy.

Claims

1. A kit for identifying lycoris radiata, which is characterized by comprising a primer combination and a probe;

the primer combination comprises a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID No.2, the nucleotide sequence of the reverse primer is shown as SEQ ID No.3, and the nucleotide sequence of the probe is shown as SEQ ID No. 4;

alternatively, the primer combination comprises three forward primers and four reverse primers, the nucleotide sequences of the forward primers are shown as SEQ ID No.2, SEQ ID No.5 and SEQ ID No.6, the nucleotide sequences of the reverse primers are shown as SEQ ID No.3, SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, and the nucleotide sequences of the probes are shown as SEQ ID No.4, SEQ ID No.10 and SEQ ID No. 11.

2. The kit for identifying lycoris mealy bugs according to claim 1, wherein the probe with the nucleotide sequence shown in SEQ ID No.4, the fluorescent group marked at the 5 'end is FAM and the quenching group marked at the 3' end is BHQ1; the nucleotide sequences of the probes are shown as SEQ ID No.10 and SEQ ID No.11, the fluorescent group marked at the 5 'end is ROX, and the quenching group marked at the 3' end is BHQ1.

3. A method for identifying lycoris radiata, characterized in that the method for identifying lycoris radiata is method (a) or method (B):

the method (A) comprises the following steps:

(a) Extracting DNA of a sample to be detected as an amplification template;

the method (B) comprises the steps of:

extracting DNA of a sample to be detected as an amplification template;

wherein, the nucleotide sequence of the probe is shown as SEQ ID No.4, the fluorescent group marked at the 5 'end is FAM, and the quenching group marked at the 3' end is BHQ1; probes with nucleotide sequences shown as SEQ ID No.10 and SEQ ID No.11, wherein a fluorescent group marked at the 5 'end is ROX, and a quenching group marked at the 3' end is BHQ1;

4. The method for identifying lycoris mealy bugs of claim 3 wherein the fluorescent quantitative PCR reaction system is:II Probe qPCR SuperMix 10. Mu.L of each of 5pmol of the primers in the primer set, 5pmol of the probe, 1. Mu.L of the template, and 20. Mu.L of the template were filled with sterile nuclease-free water; the amplification reaction conditions were 94℃for 30s of pre-denaturation; denaturation at 94℃for 5s, annealing at 61.4℃for 30s, extension at 72℃for 30s for 40 cycles.

5. The method for identifying lycoris mealy bugs of claim 3 wherein the digital PCR reaction system is: ddPCR Supermix for Probes 10. Mu.L of each of the primers in the primer set was 18pmol, 5pmol of probe, 1. Mu.L of template, and 20. Mu.L of sterile nuclease-free water; the amplification reaction condition is that the pre-denaturation is carried out for 10min at 95 ℃; denaturation at 94℃for 30s, annealing at 60℃for 60s and extension for 45 cycles; after the reaction was completed, the temperature was kept at 4 ℃.

6. The method for identifying lycoris mealy bugs of claim 3 wherein the multiplex fluorescent quantitative PCR reaction system is:II Probe qPCR SuperMix 10. Mu.L of each primer in the primer combination, 5pmol of each probe with the nucleotide sequence shown as SEQ ID No.4, 3pmol of each probe with the nucleotide sequences shown as SEQ ID No.10 and SEQ ID No.11, and 1. Mu.L of template are complemented to 20. Mu.L by sterile nuclease-free water; the amplification reaction conditions were 94℃for 30s of pre-denaturation; denaturation at 94℃for 5s, annealing at 61.4℃for 30s, extension at 72℃for 30s for 40 cycles.