阅读说明：本技术 一种检测虾肝肠胞虫的引物以及试剂盒 (Primer and kit for detecting shrimp liver enterocytozoon ) 是由 马荣荣 卢先东 钱冬 刘艳红 祝波 李小冰 于 2021-02-04 设计创作，主要内容包括：本发明涉及一种检测虾肝肠胞虫的引物以及试剂盒,所述引物具有4个,与现有技术相比,本发明具有如下优点：提供了4条不同的特异性引物,故而对虾肝肠胞虫的检测结果准确度更高,结合LAMP技术与微流控芯片技术,即可以快速给出准确的检测结果,又达到同一样品多个不同指标联检的目的,同时,反应试剂预埋于微流控芯片上,用户只需加入样品,操作简便,仪器配备锂电池,便于现场快检。(The invention relates to a primer and a kit for detecting shrimp liver enterocytozoon, wherein the number of the primer is 4, and compared with the prior art, the primer and the kit have the following advantages: the method provides 4 different specific primers, so that the detection result accuracy of the prawn enterocytozoon is higher, the LAMP technology and the microfluidic chip technology are combined, the accurate detection result can be rapidly given, the purpose of joint detection of a plurality of different indexes of the same sample is achieved, meanwhile, the reaction reagent is pre-embedded on the microfluidic chip, a user only needs to add the sample, the operation is simple and convenient, and the instrument is provided with a lithium battery, so that the on-site rapid detection is facilitated.)

1. A primer for detecting shrimp liver enterocytozoon is characterized in that: the primer is as follows:

F3：GATGCAAAGAGATATGTTGAAAG；

B3：TGCTTTAAGGTTTACAGTGTT；

FIP：CGCATTTTTCTGTACCTGTTTTGTTGAATACATTCATACAAAGTTACCAG；

BIP：GCAGAGTGTTGTTAAGGGTTTAAGTTGAAATTGTATTTGACCAATGGT。

2. a kit for detecting shrimp enterocytozoon comprising the primer of claim 1, wherein: the kit specifically comprises the following reagents:

20mM Tris-HCl pH 8.8，10mM KCl，10mM(NH₄)₂SO₄，8mM MgSO₄0.1% Tween-20, 1.4mM dNTPs; bst enzyme, 800U/mL; 50 μ M SYBRGREEN fluorescent dye; the internal reference plasmid is 400 copies/mu l; gold nanoparticles 4.0x10^-6mol/L; 0.6U FEN 1; the primer for the shrimp enterocytozoon is 90 mu M and 0.5 mu L.

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a primer and a kit for rapidly detecting shrimp liver enterocytozoon.

Background

The Enterocytozoon Hepaticae (EHP) belongs to the kingdom fungi, the family Enterocytocidaceae, the genus Enteromorpha (Enterocytozobiue), and is an obligate intracellular parasite. The parasite can infect many kinds of cultured shrimps such as Penaeus monodon (Penaeus monodon) and Litopenaeus vannamei (Penaeus vannamei), resulting in slow growth and even chronic death. Although the prevalence and infection intensity of EHP were low in early reports, the prevalence of EHP-induced Hepatopancreatic microsporidiosis (HPM) in the asian prawn farming industry has been gradually increased in recent years, causing serious economic losses in the prawn farming industry. The development of a high-efficiency detection technology which can be directly operated by shrimp farmers at the side of a pond has important significance for the prevention and control of the pathogens.

Molecular detection methods, particularly nucleic acid amplification, generally have high sensitivity and specificity. Polymerase Chain Reaction (PCR) is a traditional method for diagnosing many prawn pathogens. A variety of PCR-based methods (including one-step PCR, nested PCR, etc.) have been used for EHP detection. However, the one-step PCR technology has high requirements on detection equipment and operation environment, the operation is complicated, only 1 pair of primers is needed for reaction, the reaction is easy to be interfered, the specificity is insufficient, and the detection limit is usually 10³-10⁴Copies/. mu.L, which is not sensitive enough to detect the infection status of a small number of pathogen carriers; the nested PCR needs two pairs of primers, the specificity and the detection limit are improved, but the required detection time is correspondingly increased, and the concept of quick application in actual production is contradictory; in addition, the established Real-time fluorescent quantitative PCR (Real-time fluorescent quantitative PCR) method (including TaqMan PCR and SYBR Green I PCR) for EHP detection can realize pathogen detection quantification, but the required equipment is expensive, the operation is complex, the data reading is fussy, and the popularization in shrimp farmers is difficult; the nucleic acid isothermal amplification (LAMP) technology can amplify nucleic acid in a short time under an isothermal condition, is not interfered by conventional inhibitors in a sample, and has the advantages of simplicity, rapidness and strong specificityThe method has the characteristics of realizing on-site rapid detection without depending on any special instrument and equipment. However, the LAMP technology has advantages, because the LAMP technology is a constant temperature reaction, hot start enzyme similar to PCR is lacked, non-specific amplification is easily generated in the temperature rise stage of equipment, aerosol pollution is easily caused by open result observation, false positive is high, and the detection result is influenced.

The detection technology of a microfluidic chip (Microfluidics chip) is a technology based on the accurate operation of a microfluidic interface, and can integrate a sample detection process into a chip with a plurality of square centimeters. The purpose of molecular detection can be achieved by controlling, processing and reacting trace fluid in a micro-pipeline network of micron and submicron level. The technology overcomes the defects of the traditional detection technology, and has the characteristics of low manufacturing cost, low energy consumption and material consumption, miniaturized equipment, high analysis speed, high sensitivity, simple and automatic operation, integration and the like. The gene amplification and the detection of the product can be completed in one step, the sensitivity and the reaction time are superior to those of various detection methods based on PCR, the high-flux rapid detection of pathogenic nucleic acid can be realized, and the field daily monitoring and early diagnosis of the aquatic epidemic disease by shrimp farmers can be realized.

Disclosure of Invention

The first technical problem to be solved by the invention is to provide a primer capable of rapidly detecting the shrimp liver enterocytozoon aiming at the current situation of the prior art.

The second technical problem to be solved by the present invention is to provide a kit using the above primer, aiming at the current situation of the prior art.

The technical scheme adopted by the invention for solving the first technical problem is as follows: the primer for detecting the shrimp liver enterocytozoon is characterized in that: the primer is as follows:

F3：GATGCAAAGAGATATGTTGAAAG；

B3：TGCTTTAAGGTTTACAGTGTT；

FIP：CGCATTTTTCTGTACCTGTTTTGTTGAATACATTCATACAAAGTTACCAG；

BIP：GCAGAGTGTTGTTAAGGGTTTAAGTTGAAATTGTATTTGACCAATGGT。

in order to solve the second technical problem, the invention also provides a kit for detecting the shrimp liver enterocytozoon, which is characterized in that: the kit specifically comprises the following reagents:

20mM Tris-HCl pH 8.8，10mM KCl，10mM(NH₄)₂SO₄，8mM MgSO₄0.1% Tween-20, 1.4mM dNTPs; bst enzyme, 800U/mL; 50 μ M SYBRGREEN fluorescent dye; the internal reference plasmid is 400 copies/mu l; gold nanoparticles 4.0x10^-6mol/L; 0.6U FEN 1; the primer for the shrimp enterocytozoon is 90 mu M and 0.5 mu L.

Compared with the prior art, the invention has the following advantages: the method provides 4 different specific primers, so that the detection result accuracy of the prawn enterocytozoon is higher, the LAMP technology and the microfluidic chip technology are combined, the accurate detection result can be rapidly given, the purpose of joint detection of a plurality of different indexes of the same sample is achieved, meanwhile, the reaction reagent is pre-embedded on the microfluidic chip, a user only needs to add the sample, the operation is simple and convenient, and the instrument is provided with a lithium battery, so that the on-site rapid detection is facilitated.

Drawings

FIG. 1 is a process flow chart of the detection of the shrimp enterohepatic cytozoosis;

FIG. 2 shows plasmid 10 with shrimp liver Enteromorpha Gene fragment for detection restriction experiment in example 3 of the present invention⁵Amplification result map of copies/. mu.L;

FIG. 3 shows plasmid 10 with shrimp liver Enteromorpha Gene fragment for detection restriction experiment in example 3 of the present invention⁴Amplification result map of copies/. mu.L;

FIG. 4 shows plasmid 10 carrying a restriction test on the gene fragment of Enteromorpha hepatica according to example 3 of the present invention³Amplification result map of copies/. mu.L;

FIG. 5 shows plasmid 10 carrying a restriction test on the gene fragment of Enteromorpha hepatica according to example 3 of the present invention²Amplification result map of copies/. mu.L;

FIG. 6 shows plasmid 10 carrying a restriction test on the gene fragment of Enteromorpha hepatica according to example 3 of the present invention¹Amplification result map of copies/. mu.L;

FIG. 7 shows the detection limit of the gene fragment carrying the shrimp liver EnteromorphaExperimental plasmid 10⁰Amplification result map of copies/. mu.L;

FIG. 8 is a graph showing the amplification results of the negative control in example 3 of the present invention;

FIG. 9 is a graph showing the amplification results of the positive control in example 3 of the present invention;

FIG. 10 is a graph showing the amplification results of a positive sample in example 4 of the present invention;

FIG. 11 is a graph showing the amplification results of the negative control in example 4 of the present invention.

Detailed Description

The present invention is further illustrated by the following figures, sequence listing and examples.

Example 1

1) Primer design software is utilized to design 18S gene segment primers of the shrimp liver enterocytozoon, wherein the related primers refer to gene sequences in NCBI, and the gene sequences are numbered as follows: MH260592.1, see Seq No. 1;

2) fixing the screened specific primers at corresponding positions in the microfluidic chip for detecting the shrimp intestinal cytozoon, and packaging the chip, wherein the designed primer sequence is the primer for detecting the shrimp intestinal cytozoon.

Example 2

The DNA extraction comprises the following specific steps: when a shrimp sample is processed, putting the collected focus part into a homogenizing bag, fully and uniformly grinding, adding 2mL of physiological saline, fully and uniformly oscillating, sucking 100 mu L of physiological saline from the bag, placing the solution into a 1.5mL centrifuge tube, centrifuging for 1min, discarding supernatant, and keeping precipitate to perform the following steps:

(1) adding 500ul of lysis solution R, shaking for 10 seconds, and incubating at 80 ℃ for 5 min;

(2) centrifuging for 1min, sucking supernatant to a new centrifuge tube, adding 200uL of anhydrous ethanol, and mixing by vortex or inversion;

(3) transferring all the solution into an adsorption column, centrifuging for 30 seconds, and removing the filtrate;

(4) putting the adsorption column back into the collection tube again, adding 500uL of washing liquid C, centrifuging for 30 seconds, and removing the filtrate;

(5) putting the adsorption column back into the collection tube again, and adding 500uL of washing liquid D;

(6) centrifuging for 30 seconds, discarding the filtrate, and centrifuging for 30 seconds by placing the adsorption column back into the collection tube;

(7) putting the adsorption column into a new centrifugal tube, and adding 100uL of eluent above the center of a column membrane of the adsorption column;

(8) centrifuging for 1min to obtain eluate as nucleic acid to be detected;

(9) 15uL of nucleic acid +10uL of fluorescent isothermal amplification premix solution is sucked from 100uL into a new centrifugal tube, the centrifugal tube is vibrated for 10 seconds and centrifuged for 30 seconds, and all liquid (25uL) in the suction tube is added into a chip for detection.

Example 3

The sensitivity and detection limit of the primer for detecting the shrimp liver enterocytozoon 18S gene are as follows:

3.1 Experimental materials

3.1.1 reagents: reaction solution; diluting with sterile water to 1 × 10 respectively⁵copies/uL、1×10⁴copies/uL、1×10³copies/uL、1×10²copies/uL、1×10¹copies/uL、1×10⁰A plasmid of copies/uL with 18S gene fragment of shrimp liver enterocytozoon; negative control; a positive control;

3.1.2 Instrument: a constant temperature amplification instrument; a palm centrifuge; a pipettor;

3.1.3 detection System

Performing an experimental operation with reference to the above detection system, and then placing the same chip into an isothermal amplification apparatus for performing an experimental detection, wherein the amplification result can be shown as fig. 2, 3, 4, 5, 6, 7, 8, 9, and from the results of the detection limits of fig. 2 to 9, the results of the positive controls of fig. 2 to 5 and 9 all show typical S-shaped amplification curves, the results of fig. 7 to 8 are at the baseline position, and the results of fig. 6 are poor in repeatability, which indicates that the minimum detection limit of the detection system is 1 × 10²In the plasmid of copies/uL, the Ct is less than 20, which indicates that the sensitivity is higher and the detection speed is high.

Example 4

4. The repeatability of the shrimp liver enterocytozoon 18S gene primer is verified:

4.1.1 reagents: reaction solution; diluting with sterile water to 1 × 10 respectively⁴A plasmid with 18S gene segment of shrimp liver enterocytozoon in copies/uL; yin (kidney)And (4) performing sexual control.

4.1.2 Instrument: a constant temperature amplification instrument; a palm centrifuge; a pipettor;

4.1.3 detection System

And (3) carrying out experimental operation by referring to the detection system, putting the same chip into a constant-temperature amplification instrument for experimental detection, wherein the amplification result can be shown in figure 10, a typical S-shaped amplification curve is also presented, the negative result in figure 11 is at a base line position, the CV value of each group is less than 5%, and the repeatability is proved to meet the requirement.

Specific Ct and CV values are shown in the following table.

TABLE 1 repeatability verification of microfluidic shrimp liver enterocytozoon 18S gene detection

Example 5

Comparison by conventional PCR method

5.1 sample Collection

Collecting prawn samples from different prawn farms, and fully homogenizing hepatopancreas;

5.2 sample detection

(1) Weighing prawn hepatopancreas, homogenizing, and extracting nucleic acid according to the steps of example 2;

(2) diluting with sterile water to 1 × 10 respectively⁵copies/uL、1×10⁴copies/uL、1×10³copies/uL、1×10²copies/uL、1×10¹copies/uL、1×10⁰A plasmid of copies/uL with 18S gene fragment of shrimp liver enterocytozoon;

(3) taking the sample nucleic acid extracting solution, and performing nucleic acid determination by adopting a microfluidic detector;

(4) taking the above plasmid and nucleic acid sample solution, and using the primers

EHP-F：GATGCTTGGTGTGGGAGAA；

EHP-R：CCCCCCATCAATTTCCAACG；

Carrying out PCR verification reaction;

(5) the positive control is nucleic acid extracted from shrimp tissue homogenate containing the pathogen, and the negative control is a sterile water sample;

5.3. analysis of results

As can be seen from Table 2, the lowest detection limit of the conventional PCR prawn enterocytozoon hepatocyst gene is 1X 10³copies/uL, which shows that the microfluidic chip has higher sensitivity.

TABLE 2 comparison of microfluidic assay kit method with conventional PCR method

Note: "+" indicates positive result, and "-" indicates negative result

Analysis of reaction time

Calculating time after the DNA extraction is finished, wherein the PCR reaction process is 2 hours, electrophoresis is carried out for 40 minutes, the total time consumption is 2 hours and 40 minutes, and the total time consumption of the kit method is less than 30 minutes; the detection result analysis is combined, and the prawn intestinal cytozoon detection system disclosed by the invention is shown to greatly shorten the overall reaction time while keeping high sensitivity.

Sequence listing

<110> Ningbo love Gene science and technology Limited of Ningbo university

<120> primer and kit for detecting shrimp liver enterocytozoon

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 956

<212> DNA

<213> Enterocytozoon

<400> 1

gatttgctct atgcgggaag aataaccacg gtaacctgtg gctaagagtg tagaataagg 60

tgcaatccta ttagtttgtt ggtagtgtaa aggactacca aggcagtgat gggtaacggg 120

aaatcagggt ttgattccgg agagggagcc tgagagatgg ctcccacgtc caaggatggc 180

agcaggcgcg aaaattgtcc actcttttga gaggagacag ttatgaaacg tgagtagaag 240

ggtcgagtgt aaaaaccttg acgtgaagca attggagggc aagttttggt gccagcagcc 300

gcggtaattc caactccaag agtgtctatg gtggatgctg cagttaaagg gtccgtagtc 360

gtagatgcaa ttaaaaggtg gtgttaaaag ccattgagtt tgttgagagt agcggaacgg 420

atagggagca tggtataggt gggcaaagaa tgaaatctca agaccccacc tggaccaacg 480

gaggcgaaag cgatgctctt agacgtatct ggggatcaag gacgaaggct agagtatcga 540

aagtgattag acaccgctgt agttctagca gtaaactatg ccgacaatgc tgggtgttgc 600

gagagcgatg cttggtgtgg gagaaatctt agttttcggg ctctggggat agtacgctcg 660

caagggtgaa acttaaagcg aaattgacgg aaggacacta ccaggagtgg attgtgctgc 720

ttaatttaac tcaacgcggg aaaacttacc agggtcaagt ctatcgtaga ttggagacat 780

gaggtagaca agagtggtgc atggccgttg gaaattgatg gggcgacttt tagcttaagt 840

gctggaacca gtgagatctt ctagacaggt gttatttagg cacaggaggg agaaggcaat 900

aacaggtccg tgatgccctt agatatcctg ggcagcaagc gcaatacaat atctct 956