CN114058709A

CN114058709A - LAMP technology-based method for identifying Drosophila FARQ complex and special primer group thereof

Info

Publication number: CN114058709A
Application number: CN202010743692.4A
Authority: CN
Inventors: 李志红; 张岳
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-07-29
Filing date: 2020-07-29
Publication date: 2022-02-18
Anticipated expiration: 2040-07-29
Also published as: CN114058709B

Abstract

The invention discloses a LAMP technology-based method for identifying a Drosophila FARQ complex and a special primer group thereof. The special primer group consists of a primer 1-a primer 4; the primer 1 is a single-stranded DNA molecule shown as a sequence 1 in a sequence table; the primer 2 is a single-stranded DNA molecule shown in a sequence 2 in a sequence table; the primer 3 is a single-stranded DNA molecule shown as a sequence 3 in a sequence table; the primer 4 is a single-stranded DNA molecule shown in a sequence 4 in a sequence table. The special primer group has good specificity and short detection time, can effectively detect any kind of fruit fly in the leptomycosis FARQ complex, has the advantages of no dependence on expensive instruments, accuracy, rapidness, wide application range and the like, has guiding significance for rapidly detecting the leptomycosis FARQ complex, and provides a powerful tool for rapidly detecting the leptomycosis FARQ complex.

Description

LAMP technology-based method for identifying Drosophila FARQ complex and special primer group thereof

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a LAMP technology-based method for identifying a Drosophila FARQ complex and a special primer group thereof.

Background

The Drosophila diptera, the family of Drosophila, is widely distributed throughout the world, with about 500 species of 4,500, and of these, the economically significant species of Drosophila include 223 species of 26. The fruit fly has a wide host range, seriously affects the outward trade of fruits and vegetables while injuring the economic crops such as fruits, vegetables and the like, causes trade barrier and causes huge economic loss. The Drosophila cerealis FARQ complex belongs to Tephritidae, Drosophila cerealis, Parandrus cerealis and subgenus Pterandrus and consists of Ceratodon farinosa fascicularis, Psidium squamosatum Ceratoides Ananae, Nata Ceratitis rosa and Dianthus superbus Ceratoides Qulicii, wherein the Nata Ceratitis rosa can damage more than 90 crops of 25 families and is the most destructive trypetidae in the complex. This complex originates in the african continent, where the natura nasalis ceratis rosa and the fringed Ceratitis quilicii have invaded the mazeiss and wannwang in the indian ocean, and north america, south america and south east asia are all their highly habitats. In China, the complex is often intercepted at the port, and the need of quarantine identification, particularly rapid identification, is more urgent.

The traditional fruit fly identification work mainly depends on morphological characteristics, but the variety of fruit flies is large, the morphological difference for distinguishing different types of fruit flies is very fine and complicated, and the existence of the problems brings great obstruction to the traditional fruit fly morphological identification. The molecular biology technology is used for identifying the species of the fruit flies, mainly comprises a DNA bar code technology, a PCR technology, a restriction fragment length polymorphism technology and a gene chip technology, but needs expensive instruments and equipment, has high identification cost and is not suitable for popularization and application in basic departments.

Loop-mediated isothermal amplification (LAMP) designs 4-6 special primers aiming at 6 specific regions on a target gene, Bst DNA polymerase with strong strand displacement activity is adopted to react for about 30-60min under the constant temperature condition (60-65 ℃), and then a large amount of amplification of nucleic acid can be completed, and a large amount of stem-loop structure DNA fragments with different lengths are generated. LAMP is a simple and efficient nucleic acid isothermal amplification technology, can amplify a large number of reaction products in a short time, is rapid, efficient and high in sensitivity, is simple and convenient to operate, does not need special instruments, can be used for observing and judging detection results through a color reaction naked eye, and has better application and popularization prospects.

Disclosure of Invention

The invention aims to provide a LAMP technology-based method for identifying a Drosophila FARQ complex and a special primer group thereof.

In a first aspect, the invention features a set of primer sets for detecting or aiding in the detection of the Drosophila FARQ complex.

The complete set of primer group for detecting or assisting in detecting the Serratia FARQ complex provided by the invention consists of a primer 1-a primer 4;

the primer 1 is a1) or a2) as follows:

a1) a single-stranded DNA molecule shown as a sequence 1 in a sequence table;

a2) a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1;

the primer 2 is a3) or a4) as follows:

a3) a single-stranded DNA molecule shown in a sequence 2 in a sequence table;

a4) a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2;

the primer 3 is a5) or a6) as follows:

a5) a single-stranded DNA molecule shown in a sequence 3 in a sequence table;

a6) a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3;

the primer 4 is a7) or a8) as follows:

a7) a single-stranded DNA molecule shown in a sequence 4 in a sequence table;

a8) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 4 and having the same function as the sequence 4.

In the above primer set, the molar ratio of the primer 1, the primer 2, the primer 3 and the primer 4 is 1:1:8: 8.

In a second aspect, the invention protects a new use of the primer set.

The invention protects the application of the primer set in any one of the following b1) -b 6):

b1) preparing a product for detecting or assisting in detecting the Serratia FARQ complex;

b2) detecting or assisting in detecting the Serratia FARQ complex;

b3) preparing a product for detecting or assisting in detecting whether the fruit fly to be detected belongs to a Serratula FARQ complex;

b4) detecting or detecting in an auxiliary way whether the fruit fly to be detected belongs to a Serratula FARQ complex;

b5) preparing products for identifying or assisting in identifying the Scleroderma FARQ complex and other fruit flies;

b6) identifying or assisting in identifying the Scleroderma FARQ complex and other fruit flies.

In a third aspect, the present invention provides a kit comprising the above primer set;

the function of the kit is any one of the following c1) -c 3):

c1) detecting or assisting in detecting the Serratia FARQ complex;

c2) detecting or detecting in an auxiliary way whether the fruit fly to be detected belongs to a Serratula FARQ complex;

c3) identifying or assisting in identifying the Scleroderma FARQ complex and other fruit flies.

Further, the kit may also include other reagents for detecting the Drosophila FARQ complex. In the present invention, the other reagent for detecting the Serratia FARQ complex is the Water Start LAMP2 × Master Mix.

Still further, the kit may further comprise sterile ultrapure water.

In a fourth aspect, the invention provides a method for preparing the kit. The preparation method of the kit comprises the following steps of d1) or d 2):

d1) packaging each primer in the primer set separately;

d2) and mixing the primers in the primer set according to the proportion.

In the preparation method of the kit, in the d2), the primer 1, the primer 2, the primer 3 and the primer 4 in the primer set are mixed together according to a molar ratio of 1:1:8: 8.

In a fifth aspect, the invention provides a method for detecting or assisting in detecting whether a fruit fly to be detected belongs to a Serratia FARQ complex.

The method for detecting or assisting in detecting whether the fruit fly to be detected belongs to a Scomberous FARQ complex or not comprises the following steps: extracting the nucleic acid of the fruit fly to be detected, and performing loop-mediated isothermal amplification by using the whole set of primer set by using the nucleic acid of the fruit fly to be detected as a template; after the loop-mediated isothermal amplification reaction is finished, whether the fruit fly to be detected belongs to a tripdioides FARQ complex is judged by detecting whether a step-shaped band appears after electrophoresis of a reaction product: if the reaction product presents a step-shaped strip after electrophoresis, the fruit fly to be detected belongs to or is a candidate of a small-strip fruit fly FARQ complex; if the reaction product has no stepped strip after electrophoresis, the fruit fly to be detected does not belong to or candidate does not belong to the small-strip fruit fly FARQ complex.

In a sixth aspect, the invention features a method of identifying or aiding in identifying the Serratia FARQ complex from other fruit flies.

The method for identifying or assisting in identifying the Scleroderma FARQ complex and other fruit flies comprises the following steps: extracting the nucleic acid of the fruit fly to be detected, taking the nucleic acid of the fruit fly to be detected as a template, adopting the complete set of primer set to carry out loop-mediated isothermal amplification, and judging whether the fruit fly to be detected belongs to a small-striped fly FARQ complex or other fruit flies by detecting whether a step-shaped strip is formed after electrophoresis of a reaction product after the loop-mediated isothermal amplification reaction is finished: if the reaction product presents a step-shaped strip after electrophoresis, the fruit fly to be detected belongs to or is a candidate of a small-strip fruit fly FARQ complex; if the reaction product has no stepped band after electrophoresis, the fruit fly to be detected is or is selected as other fruit flies.

In any of the above methods, the nucleic acid is genomic DNA.

The loop-mediated isothermal amplification reaction system comprises the following components: mu.L of Water Start LAMP2 × Master Mix, 1. mu.L of LDNA template, 2.5. mu.L of sterile ultrapure water, and 9. mu.L of primer Mix. The final concentration of the primer 1 and the final concentration of the primer 2 in the primer mixture in a reaction system are both 0.2 mu M; the final concentration of the primer 3 and the final concentration of the primer 4 in the reaction system are both 1.6 mu M.

The loop-mediated isothermal amplification reaction conditions are as follows: the reaction was carried out at 65 ℃ for 40 min.

In any of the above-described uses or kits or methods, the other fruit fly may be at least one of the following fruit flies: blackberry fruit fly Ceratitis rubivora, African mango fruit fly Ceratitis cosyla, Mediterranean fruit fly Ceratitis capita, five-spotted fruit fly Ceratitis quinaria.

In any of the uses or kits or methods described above, the drosophila FARQ complex consists of drosophila ventricosa cerasus, custara squamosa, bactrocera narcisternae rosae, bactrocera natsudana rosa and bactrocera superba ceralis quilegia.

The invention provides a method for identifying a Drosophila FARQ complex and a special primer group thereof based on LAMP technology. Experiments prove that: the special primer group has good specificity and short detection time, and can effectively detect any kind of fruit fly in the Serratia FARQ complex. The method has the advantages of no dependence on expensive instruments, accuracy, rapidness, wide application range and the like, has guiding significance for rapid detection of the fasciola FARQ complex, and provides a powerful tool for rapid detection of the fasciola FARQ complex.

Drawings

FIG. 1 shows the results of the primer set FARQ1-FARQ10 screening in example 2.

FIG. 2 shows the results of the specificity test in example 3.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The extraction kit for genomic DNA from a fruit fly sample in the following examples is a product of Tiangen corporation, catalog No. DP 304. Water Start LAMP2 × Master Mix is a product from NEW ENGLAND Biolabs, catalog number E1700S.

The Drosophila FARQ complexes in the examples below consisted of the small fasciola ventricosa Ceratopteris fascicularis, the fruit fly Anneae Annaeus, the fruit fly Ceratopteris rosa and the small Dianthus Ceratopteris Qulicii.

The following examples of smaller belly-spotted fruit flies Ceratopteris fascicularis, Annona squamosa fruit flies Ceratopteris anoane, Natalus fruit flies Ceratopteris Rosa, Dianthus small fruit flies Ceratopteris Qulicii, Rubus blackberry fruit flies Ceratoptera rubivora, African mango fruit flies Ceratoptera cosyra, Mediterranean fruit flies Ceratoptera capitata and five-spotted fruit flies Ceratopteris quinaria are described in the literature: wujiazui, the identification atlas of important insects of trypetid class [ M ]. Guangdong science and technology Press, 2009.

Example 1 design of primer set for identifying Drosophila FARQ Complex

The total length sequences of COI and Cytb genes in the mitochondrial genome of the fasciola FARQ complex (the total length sequences of COI genes of the fasciola ventricosus, the fruit fly Ceratitis anhoane, the fruit fly Ceratitis rostis rosa and the fruit fly Ceratitis Qulicii are respectively shown in SEQ ID No. 5-8; the total length sequences of Cyi genes of the fasciola ventricosus, the fruit fly Ceratitis faratitis fascialis, the fruit fly Ceratitis annona anhoane, the fruit fly Ceratitis nasalis and the fruit fly Quickis respectively shown in SEQ ID No. 9-12) are used as target genes, and a primer group FARQ 395, a primer group FARQ2, a primer group 3, a primer group 3563, a FARQ primer group 35 5, a FARQ primer group 3646 and a FARQ 358626 are synthesized to identify the fasciola FARQ complex. The primer sequences of the respective primer sets are detailed in Table 1.

TABLE 1 primer sequences of the respective primer sets

Example 2 screening of primer set for identifying Drosophila FARQ Complex

1. And extracting the genome DNA of the fruit fly sample by adopting a genome DNA extraction kit to obtain the genome DNA of the fruit fly sample.

The fruit fly samples are small belly spot fruit fly Ceratitis fascicularis, Nataler fruit fly Ceratitis rosa, blackberry fruit fly Ceratitis rubivora. All test fruit fly samples were identified using morphological methods prior to testing and multiplexed using DNA barcode technology.

2. And (3) performing loop-mediated isothermal amplification by using genome DNA of a fruit fly sample as a template and adopting a primer group FARQ1, a primer group FARQ2, a primer group FARQ3, a primer group FARQ4, a primer group FARQ5, a primer group FARQ6, a primer group FARQ7, a primer group FARQ8, a primer group FARQ9 and a primer group FARQ10 respectively to obtain amplification products.

The reaction system was 25. mu.L, consisting of 12.5. mu.L of Water Start LAMP2 × Master Mix, 1. mu.L of DNA template, 2.5. mu.L of sterile ultrapure water and 9. mu.L of primer Mix (10. mu. M F30.5.5. mu.L, 10. mu. M B30.5.5. mu.L, 10. mu.M FIP 4. mu.L, 10. mu.M BIP 4. mu.L). The primer mixture is a mixture of each primer in the primer group. In the reaction system, the final concentrations of the outer primer F3 and the outer primer B3 in the reaction system are both 0.2. mu.M, and the final concentrations of the inner primer FIP and the inner primer BIP in the reaction system are both 1.6. mu.M.

Reaction conditions are as follows: 40min at 65 ℃.

3. The amplification product was subjected to 2% agarose gel electrophoresis.

The detection results of the primer set FARQ1, the primer set FARQ2, the primer set FARQ3, the primer set FARQ4, the primer set FARQ 387 5, the primer set FARQ6, the primer set FARQ7, the primer set FARQ8, the primer set FARQ9 and the primer set FARQ10 are shown in FIG. 1, wherein lanes 1-3 are the primer set FARQ1, lanes 4-6 are the primer set FARQ2, lanes 7-9 are the primer set FARQ3, lanes 10-12 are the primer set FARQ4, lanes 13-15 are the primer set FARQ5, lanes 16-18 are the primer set FARQ6, lanes 19-21 are the primer set FARQ7, lanes 22-24 are the primer set FARQ8, lanes 25-27 are the primer set FARQ9, and lanes 28-30 are the primer set FARQ 10; m is DNA Marker, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28 is the small strip fruit fly Ceratitis fascicularis, 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 is the Netar fruit fly Ceratitis rosa, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 is the blackberry fruit fly Ceratitis rubivora.

The results show that primer set FARQ10 shows a clear and bright typical ladder-like band when identifying the small-stringy fly FARQ complex, and no band appears in the negative control, compared with other primer sets. Thus, the primer set FARQ10 was used to identify the Drosophila FARQ complex for the specificity experiments described below.

Example 3 specificity test

The fruit fly 1 to be tested is the small fruit fly Ceratitis fascicularis with the number of 1 in the table 2.

The fruit fly 2 to be tested is the fruit fly Ceratitis anoane numbered 2 in table 2.

The fruit fly 3 to be tested is the Nataler fruit fly Ceratitis rosa numbered 3 in Table 2.

The fruit fly 4 to be tested is Dianthus superbus Ceratitis quiciii numbered 4 in Table 2.

The fruit fly 5 to be tested is Dianthus superbus Ceratitis quiciii numbered 5 in Table 2.

The fruit fly 6 to be tested is Dianthus superbus Ceratitis quiciii numbered 6 in Table 2.

The fruit fly 7 to be tested is blackberry fruit fly Ceratitis rubivora numbered 7 in Table 2.

The fruit fly 8 to be tested is the African mango fruit fly Ceratitis cosyla numbered 8 in Table 2.

The fruit fly 9 to be tested is the African mango fruit fly Ceratitis cosyla numbered 9 in Table 2.

The fruit fly 10 to be tested is the African mango fruit fly Ceratitis cosyla numbered 10 in Table 2.

The fruit fly 11 to be tested is the African mango fruit fly Ceratitis cosyla numbered 11 in Table 2.

The fruit fly 12 to be tested is Mediterranean fruit fly Ceratitis capitata numbered 12 in Table 2.

The fruit fly 13 to be tested is Mediterranean fruit fly Ceratitis capitata numbered 13 in Table 2.

The fruit fly 14 to be tested is the five-spotted fruit fly Ceratitis quinaria with the number of 14 in Table 2.

The fruit fly 15 to be tested is the five-spotted fruit fly Ceratitis quinaria with the number of 15 in Table 2.

All test fruit fly samples were identified using morphological methods prior to testing and multiplexed using DNA barcode technology.

TABLE 2 sample number, seed name and geographical population information of test fruit fly

The method comprises the following steps of:

1. and extracting the genome DNA of the fruit fly to be detected by adopting a genome DNA extraction kit.

2. And (3) taking the genome DNA of the fruit fly to be detected extracted in the step (1) as a template, and performing loop-mediated isothermal amplification by adopting a primer group FARQ10 to obtain an amplification product.

Reaction conditions are as follows: 40min at 65 ℃.

3. The amplification product was subjected to 2% agarose gel electrophoresis.

The DNA template was replaced with sterile ultrapure water as described above, and the remaining steps were unchanged as a negative control.

The detection result of the primer set FARQ10 is shown in FIG. 2(M is DNA Marker, 1 is Ceratodon ventralis fascicularis, 2 is Annona squamosa, 3 is Netar fruit fly Ceratodon rosaceus, 4-6 are Dianthus small fruit fly Ceratoides, 7 is blackberry fruit fly Ceratodon rubivora, 8-11 are African mango fruit fly Ceratodon cosyla, 12-13 are Mediterrata Ceratoides, 14-15 are five-point fruit fly Ceratonis quinaria, N is negative control).

The result shows that the fruit fly to be detected presents a step-shaped strip when the fruit fly is a small-strip fruit fly FARQ complex; when the fruit fly to be detected is a non-fasciculus fruit fly FARQ complex, no step-shaped strip (no strip) exists; the negative control also had no bands.

Therefore, the primer group FARQ10 is adopted to identify the small-strip fly FARQ complex with good specificity.

In practical application, whether the fruit fly to be detected belongs to the tripdioides FARQ complex or not can be detected or assisted to be detected by the following method: extracting the nucleic acid of the fruit fly to be detected, and performing loop-mediated isothermal amplification by using the nucleic acid of the fruit fly to be detected as a template and adopting a primer group FARQ10 in the embodiment 1; after the loop-mediated isothermal amplification reaction is finished, whether the fruit fly to be detected belongs to a tripdioides FARQ complex is judged by detecting whether a step-shaped band appears after electrophoresis of a reaction product: if the reaction product presents a step-shaped strip after electrophoresis, the fruit fly to be detected belongs to or is a candidate of a small-strip fruit fly FARQ complex; if the reaction product has no stepped strip after electrophoresis, the fruit fly to be detected does not belong to or candidate does not belong to the small-strip fruit fly FARQ complex.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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tgacttgcca cacttcatgg tacacaatta aactactcac cagctatatt atgagctctt 1020

ggatttgtat ttttatttac agttggaggt ttaacaggag ttgttcttgc taattcttct 1080

gttgatatta ttttacacga cacttattat gttgtagcac acttccatta tgttttatct 1140

ataggagcag tatttgctat tatagctgga tttgttcact ggtatccatt atttactgga 1200

ttaaatttaa accctaaatg attaaaaagt caatttatta ttatatttat tggagtaaat 1260

ttaacatttt tccctcaaca tttcttagga ttagctggaa tacctcgccg atattctgat 1320

tatccagacg cttatacaac ttgaaatgta atttcaacaa ttggttcttc aatttctcta 1380

ttaggaattt tattcttttt atttattatt tgagaaagta taatttctca acgtcaagtt 1440

atttacccaa tacatttaaa ttcttcaatt gaatgacttc aaaatactcc acctgctgaa 1500

catagttatt ctgaactacc tcttttaact aattaa 1536

<210>8

<211>1536

<212>DNA

<213>Artificial Sequence

<400>8

tcgcaacaat ggctattttc aactaatcat aaagatattg gaactttata ttttattttt 60

ggagcatgag ctggaatagt aggaacatct cttagaattt taatccgagc tgaattaggt 120

cacccaggag cactaattgg agatgatcaa atttataatg taattgttac tgctcatgct 180

ttcgtaataa ttttttttat agttatacct attataattg gaggatttgg aaattgacta 240

gtacctttaa tacttggtgc tccagatata gcattccctc gaataaataa tataagtttt 300

tgattattac ccccttctct tacattatta ttagtaagta gtatagtaga aaatggagct 360

ggaacaggtt gaacagttta ccctcccctt tcttctgtaa ttgcccatgg aggagcttct 420

gttgacttag caattttttc tcttcactta gcaggaattt cttctatttt aggagctgta 480

aattttatta ctacagtaat taatatacga tcaactggaa tttcatttga ccgtatacct 540

ttatttgttt gagctgtagt tcttactgca ttattattat tactttctct tccagtttta 600

gctggagcta tcacaatatt attaacagat cgaaatttaa atacttcatt ctttgatcca 660

gcaggaggag gggatcctat tttataccaa cacttatttt gattttttgg acatcctgaa 720

gtatatattt taattctacc tggattcgga ataatttctc atattattag tcaagaatca 780

ggaaaaaagg aaacattcgg atcattagga ataatttatg ctatattagc aattggttta 840

ttaggattta ttgtttgagc acatcatata tttacagttg gtatagatgt agatactcga 900

gcttatttta catcagcaac aataattatt gctgttccaa caggtattaa aatttttagt 960

tgacttgcca cacttcatgg tacacaatta aactactcac cagctatatt atgagccctt 1020

ggatttgtat ttttatttac agttggaggt ttaacaggag ttgttcttgc taattcttct 1080

gttgatatta ttttacacga tacttattat gttgtagcac acttccatta tgttttatct 1140

ataggagcag tatttgctat tatagctgga tttgttcact gatatccatt atttactgga 1200

ttaaatttaa atcctaaatg attaaaaagt caatttatta ttatatttat tggagtaaat 1260

ttaacatttt tccctcaaca ttttttagga ttagctggaa tacctcgtcg atattcagat 1320

tatccagatg cttatacaac ttgaaatgta atttcaacaa ttggttcttc aatttctctg 1380

ttaggaattt tattcttttt atttattatt tgagaaagta taatttctca acgtcaagtt 1440

atttacccaa tacatttaaa ttcttcaatt gaatgacttc aaaatactcc acctgctgaa 1500

catagttatt ctgaactacc tcttttaact aattaa 1536

<210>9

<211>1137

<212>DNA

<213>Artificial Sequence

<400>9

atgaacaaac ctttacgaac ccaacacccc ttatttaaaa ttgctaataa tgctttagta 60

gatttacctg cacctattaa tatctcagct tgatgaaatt ttggttcatt attaggatta 120

tgtttaatta tccaaatttt aactggatta ttccttgcta tacattacac agcagatatt 180

aatttagcct tcaatagagt taatcatatt tgtcgagatg taaattatgg atgattatta 240

cgaactatac acgctaacgg tgcatcattc ttttttattt gtatttattt acatgtagga 300

cgtggaatrt attatggatc ttatttattc accccaacat gattaattgg agtattaatt 360

ttatttttag taatagcaac agcttttata ggttatgttc ttccatgagg acaaatatca 420

ttttgaggag ctacagttat tactaattta ttatcagcta tcccttattt aggaattgat 480

ttagttcaat gagtatgagg aggatttgca gttgacaatg ctactttaac tcgattcttc 540

acttttcatt ttattctccc atttattgta ttagctataa ctttaattca tctattattt 600

ttacatcaaa ctggatctaa taacccaatt ggtattaatt ctaatattga taaaattcca 660

ttccaccctt attttacatt caaggatatt gttggattta ttattataat tatagcactt 720

ctcttattaa ctttaattaa tccttattta ctaggagatc cagataattt tattccagca 780

aatcctttag ttactccagt ccatattcaa cctgaatgat actttttatt tgcatatgct 840

attttacgat caattccaaa taaattagga ggagtaattg cccttgtttt atcaattgca 900

attttagcta ttttaccatt ttataattta agaaaattcc gaggaattca attttatcct 960

attaataaaa ttttattttg aattatagta gtaacagtaa ttttattaac atgaattgga 1020

gcacgaccag ttgaagaacc ttacgtttta acaggtcaaa ttttaacagt agtttatttc 1080

ttatattata tcattaaccc acttgttaat aaatgatgag acaacttaat taactag 1137

<210>10

<211>1137

<212>DNA

<213>Artificial Sequence

<400>10

atgaacaaac ctttacgaac ccaacacccc ttatttaaaa ttgctaataa tgctttagta 60

gatttacctg cacctattaa tatctcagct tgatgaaatt ttggttcatt attaggatta 120

tgtttaatta tccaaatttt aactggatta ttccttgcta tacattacac agcagatatt 180

aatttagcct tcaatagagt taatcatatt tgtcgagatg taaattatgg atgattatta 240

cgaactatac acgctaacgg tgcatcattc ttttttattt gtatttattt acatgtagga 300

cgtggaatgt attatggatc ttatttattc accccaacat gattaattgg rgtattaatt 360

ttatttttag taatagcaac agcttttata ggttatgttc ttccatgagg acaaatatca 420

ttttgaggag ctacagttat tactaattta ttatcagcta tcccttattt aggaattgat 480

ttagttcaat gagtatgagg aggatttgca gttgacaatg ctactttaac tcgattcttc 540

acttttcatt ttattctccc atttattgta ttagctataa ctttaattca tctattattt 600

ttacatcaaa ctgggtctaa taacccaatt ggtattaatt ctaatattga taaaattcca 660

ttccaccctt attttacatt caaggatatt gttggattta ttattataat tatagcactt 720

ctcttattaa ctttaattaa tccttattta ctaggagatc crgataattt tattccagca 780

aatcctttag ttactccagt ccatattcaa cctgaatgat actttttatt tgcatatgct 840

attttacgat caattccaaa taaattagga ggagtaattg cccttgtttt atcaattgca 900

attttagcya ttttaccatt ttataattta agaaaattcc gaggaattca attttatcct 960

atcaataaaa ttttattttg aattatagta gtaacagtaa ttttattaac atgaattgga 1020

gcacgaccag ttgaagaacc ttacgtttta acaggtcaaa ttttaacagt agtttatttc 1080

ttatattata tcattaaccc acttgttaat aaatgatgag acaacttaat taactag 1137

<210>11

<211>1137

<212>DNA

<213>Artificial Sequence

<400>11

atgaacaaac ctttacgaac ccaacaccct ttattcaaaa ttgctaataa tgctttagta 60

gatttacctg cacctattaa tatttcagct tgatgaaatt ttggttcatt attaggatta 120

tgtttaatta tccaaatttt aactggatta ttccttgcta tacattacac agcagatatt 180

aatttagctt ttaatagagt taatcatatt tgtcgagatg taaattatgg atgattatta 240

cgaactatac acgctaacgg tgcatcattc ttttttattt gtatttattt acatgtagga 300

cgtggaattt attacggatc ttatttattc accccaacat gattaattgg agtattaatt 360

ttatttttag taatagcaac agctttcata ggttatgttc ttccatgagg acaaatatca 420

ttttgaggag ctacagttat tactaattta ttatcagcta tcccttattt aggaattgat 480

ttagttcaat gagtatgagg aggatttgca gttgacaatg ctactttaac tcgattcttt 540

acttttcatt ttatcctccc atttattgtt ttagctataa ctttaattca ccttttattt 600

ttacatcaaa ctggatctaa taacccaatt ggtattaatt ctaatattga taaaattcca 660

ttccatcctt attttacatt caaggatatt gttggattta tcattataat tatagctctt 720

ctcttattaa ctttaattaa tccttattta ttaggagacc cagataattt tattccagca 780

aatcctttag ttactccagt ccacattcaa cctgaatgat actttttatt tgcatatgct 840

attttacgat caattcccaa taagttagga ggagtaattg cccttgtttt atcaattgca 900

attttagcta ttttaccatt ttataattta agaaaattcc gaggaattca attttatcct 960

attaataaaa ttttattctg aattatagta gtaacagtaa ttttattaac atgaattgga 1020

gcacgaccag ttgaagaacc ttacgtttta acaggtcaaa ttttaacagt aatttatttc 1080

ttatattata ttattaaccc acttattaat aaatgatgag ataacttaat taactag 1137

<210>12

<211>1137

<212>DNA

<213>Artificial Sequence

<400>12

atgaacaaac ctttacgaac ccaacacccc ttatttaaaa ttgctaataa tgctttagta 60

gatttacctg cacctattaa tatctcagct tgatgaaatt ttggttcatt attaggatta 120

tgtttaatta tccaaatttt aactggatta tttcttgcta tacattatac agcagatatt 180

aatttagctt tcaatagagt taatcatatt tgtcgagatg taaattatgg atgattatta 240

cgaactatac acgctaacgg tgcatcattc ttttttattt gtatttattt acatgtagga 300

cgtggaatgt attatggatc ttatttattc accccaacat gattaattgg agtattaatt 360

ttatttttag taatagcaac agcttttata ggttatgttc ttccatgagg acaaatatca 420

ttttgaggag ctacagttat tactaattta ttatcagcta tcccttattt aggaattgat 480

ttagttcaat gagtatgagg aggatttgca gttgacaatg ctactttaac tcgattcttc 540

acttttcatt ttattctccc atttattgta ttagctataa ctttaattca tctattattc 600

ttacatcaaa ctggatctaa taatccaatt ggtattaatt ctaatattga taaaattcca 660

tttcaccctt attttacatt caaggatatt gttggattta ttattataat tatagcactt 720

ctcttattaa ctttaattaa tccttattta ttaggagacc cagataattt tattccagca 780

aatcctttag ttaccccagt ccacattcaa cctgaatgat actttttatt tgcatatgct 840

attttacgat caattccaaa taaattagga ggagtaattg cccttgtttt atcaattgca 900

attttagcta ttttaccatt ttataattta agaaaattcc gaggaattca attttatcct 960

attaataaaa ttttattttg aattatagta gtaacagtaa ttttattaac atgaattgga 1020

gcacgaccag ttgaagaacc ttacgtttta acaggtcaaa ttttaacagt agtttatttc 1080

ttatattata tcattaaccc acttgttaat aaatgatgag ataacttaat taactag 1137

Claims

1. The complete set of primer group for detecting or assisting in detecting the Serratia FARQ complex consists of a primer 1-a primer 4;

the primer 1 is a1) or a2) as follows:

a1) a single-stranded DNA molecule shown as a sequence 1 in a sequence table;

the primer 2 is a3) or a4) as follows:

a3) a single-stranded DNA molecule shown in a sequence 2 in a sequence table;

the primer 3 is a5) or a6) as follows:

a5) a single-stranded DNA molecule shown in a sequence 3 in a sequence table;

the primer 4 is a7) or a8) as follows:

a7) a single-stranded DNA molecule shown in a sequence 4 in a sequence table;

2. The set of primers of claim 1, wherein: the molar ratio of the primer 1 to the primer 2 to the primer 3 to the primer 4 is 1:1:8: 8.

3. The use of the primer set of claim 1 or 2 in any one of the following b1) -b 6):

b2) detecting or assisting in detecting the Serratia FARQ complex;

4. A kit comprising the primer set according to claim 1 or 2;

the function of the kit is any one of the following c1) -c 3):

c1) detecting or assisting in detecting the Serratia FARQ complex;

5. The method for preparing the kit according to claim 4, wherein the kit is d1) or d 2):

d1) packaging each primer in the primer set of claim 1 or 2 separately;

d2) the primer sets of claim 1 or 2, wherein the primers are mixed together in proportion.

6. The method of claim 5, wherein: in the d2), the primer 1, the primer 2, the primer 3 and the primer 4 in the primer set are mixed together according to a molar ratio of 1:1:8: 8.

7. A method for detecting or assisting in detecting whether fruit flies to be detected belong to a Serratia FARQ complex or not comprises the following steps: extracting the nucleic acid of the fruit fly to be detected, and performing loop-mediated isothermal amplification by using the set of primer group of claim 1 by using the nucleic acid of the fruit fly to be detected as a template; after the loop-mediated isothermal amplification reaction is finished, whether the fruit fly to be detected belongs to a tripdioides FARQ complex is judged by detecting whether a step-shaped band appears after electrophoresis of a reaction product: if the reaction product presents a step-shaped strip after electrophoresis, the fruit fly to be detected belongs to or is a candidate of a small-strip fruit fly FARQ complex; if the reaction product has no stepped strip after electrophoresis, the fruit fly to be detected does not belong to or candidate does not belong to the small-strip fruit fly FARQ complex.

8. A method for identifying or assisting in identifying a Scleroderma FARQ complex from other fruit flies, comprising the steps of: extracting the nucleic acid of the fruit fly to be detected, taking the nucleic acid of the fruit fly to be detected as a template, adopting the primer set of claim 1 to carry out loop-mediated isothermal amplification, and judging whether the fruit fly to be detected belongs to a tripdioides FARQ complex or other fruit flies by detecting whether a stepped strip is presented after electrophoresis of a reaction product after the loop-mediated isothermal amplification reaction is finished: if the reaction product presents a step-shaped strip after electrophoresis, the fruit fly to be detected belongs to or is a candidate of a small-strip fruit fly FARQ complex; if the reaction product has no stepped band after electrophoresis, the fruit fly to be detected is or is selected as other fruit flies.

9. The method according to claim 7 or 8, characterized in that: the final concentration of the primer 1 and the final concentration of the primer 2 in the reaction system are both 0.2 mu M, and the final concentration of the primer 3 and the final concentration of the primer 4 in the reaction system are both 1.6 mu M;

or, the reaction conditions of the loop-mediated isothermal amplification are as follows: the reaction was carried out at 65 ℃ for 40 min.

10. The set of primers of claim 1 or 2 or the use of claim 3 or the kit of claim 4 or the method of any one of claims 7 to 9, wherein: the Drosophila FARQ complex consists of the small fasciola ventricosa cerasus Ceratitis fascicularis, the Annona squamosa cerana cerasus, the Netar cerasus rosa and the Dianthus superbus cerasus Ceratitis quinciii.