CN113512590A - Kit for detecting different echinococcus species based on honeycomb chip and detection method - Google Patents
Kit for detecting different echinococcus species based on honeycomb chip and detection method Download PDFInfo
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Abstract
The invention discloses a honeycomb chip-based detection kit for detecting different echinococcosis species, which is used for simultaneously detecting echinococcosis granulosa and echinococcosis multilocularis, and 2 insect strains in total. The kit comprises the following primers: echinococcus granulosus primers, 4, SEQ ID No. 1-4; echinococcus multilocularis primer, 4 pieces, SEQ ID NO. 5-8. In addition, the invention also discloses a method for detecting different echinococcus species based on the honeycomb chip. Various verifications show that the kit has the characteristics of rapidness, sensitivity and strong specificity, solves the problem that the basic layer does not have the complicated steps of PCR instrument detection and electrophoresis, can simultaneously detect a plurality of genes by one-time reaction, defines the insect species of the insect species, greatly lightens the workload, shortens the detection time, reduces the detection cost and has greater development tendency.
Description
Technical Field
The invention relates to the technical field of agriculture and animal quarantine, in particular to an LAMP (loop-mediated isothermal amplification) detection method for two echinococcosis, and particularly relates to a honeycomb chip-based kit and a detection method for detecting different echinococcosis species. Furthermore, the invention relates to specific primers for the detection of different species of echinococcus.
Background
Echinococcosis (Echinococcosis), also known as Echinococcosis (Hydatidosis), is a serious zoonotic parasitic disease caused by the parasitosis of Echinococcus larvae in humans and various animals. Has become a global public health problem, with costs as much as 30 billion dollars annually for echinococcosis treatment and animal husbandry losses. With the progress of globalization of the animal and animal product trade, the disease has a tendency to spread from the northern hemisphere to the southern hemisphere. China is one of the countries with the highest incidence rate of echinococcosis, 31 provinces, cities and autonomous regions have reports of the echinococcosis, wherein echinococcosis is prevalent in western regions such as inner Mongolia, Xinjiang, Gansu, Ningxia, Qinghai, Tibet, northwest Sichuan, Shaanxi and other pasturing regions and semi-pasturing regions, the high incidence region accounts for about 44% of the area of the whole country, and the threatened population accounts for about 5000 ten thousand. At the professional meeting of parasites in western regions of China held in 2000, echinococcosis was identified as the most serious parasitic disease that endangers the development of western regions and the health of people. For example, the mean infection rate of echinococcus ovis in inner Mongolia region is 14.92% from 1990 to 1992; the monitoring point of 1991 and 1995 detects that the average positive infection rate of 3982 people is 9.82% in serology, and the annual cases are in an increasing trend. The economic loss of human and livestock caused by echinococcosis in China accounts for about 40 percent of the world, is the first place in the world and is not effectively controlled so far.
There are currently 5 independent species recognized, echinococcus granulosus tapeworm (ii) ((iii))Echinococcus granulosus Eg), Echinococcus multilocularis: (Echinococcus multilocularisEm), Echinococcus shikoensis, Echinococcus olitorius, and Echinococcus vomerozoensis larvae. The epidemic Eg and Em in China mainly account for 90% of the infusorian cases, wherein the Eg is particularly serious in harm and mainly prevails in northwest herdingIn districts and semi-farming and semi-pasturing areas, domestic dogs are the major source of infection and the ultimate host (echinococcosis); em is highly lethal and has a small distribution range, and is often found in parts of Qinghai, Tibet, Gansu, Sichuan, Xinjiang and Ningxia. Echinococcus spinosus discharges mature segments and a large amount of ova through an end host (dog, wolf, fox and the like), can pollute grassland, water source and household environment or is attached to fur of the Echinococcus spinosus to infect intermediate hosts (human, cattle, sheep and the like), so that the ova hatch in small intestines, develop into hexacoocaria which enters the whole body along with blood circulation and mainly stays in liver, lung and abdominal cavity to form echinococcus spinosus sacs, and cause the occupational organ space occupying mechanical damage which is far more serious than the intestinal symptoms of the end host. These are important factors for the disease causing poverty and the disease returning poverty in western farming and pasturing areas, and the prevention and control of the diseases also face serious challenges. As the population moves and domesticated pets increase, echinococcosis has a tendency to spread in the eastern middle cities. The disease can persist in the final host for several years. Therefore, the method for rapidly detecting the echinococcosis in the echinococcosis epidemic area has urgent practical value, is beneficial to timely finding the potential infected final host, constructs good animal husbandry development and plays an important role in prevention and control.
At present, the research of the method still adopts serology and morphology detection in China, so that the method is easy to miss detection and false detection. The lack of rapid detection tools, no detection kit and no high-throughput detection technology seriously restrict the monitoring and research on the echinococcosis.
With the development of molecular biology technology, the detection and identification of 2 types of hydatid can be realized based on different molecular markers. However, the detection of a single insect species is expensive, time-consuming and labor-consuming. A novel isothermal Nucleic acid amplification (LAMP) technique suitable for gene diagnosis was disclosed in journal of Nucleic Acids Res by Notomi, a Japanese scholarly in 2000. The technology has been successfully applied to the detection of diseases such as SARS, avian influenza, HIV, H1N1 influenza and the like, and is also widely applied to the detection of diseases caused by various viruses, bacteria, parasites and the like in Japan, the safety inspection of food and cosmetics and the rapid diagnosis of import and export. The technology has the advantages of high specificity and high sensitivity, is very simple to operate, has low requirements on instruments and equipment, can realize reaction by one water bath kettle or thermostat, can judge the result by observing the generation of white turbidity or green fluorescence by naked eyes, and is suitable for rapid diagnosis of basic layers. Is a novel nucleic acid amplification method, and is characterized in that 4 specific primers are designed aiming at 6 regions of a target gene, under the action of strand displacement DNA polymerase (Bst DNA polymerase), the nucleic acid amplification of 10^9 to 10^10 times can be realized within 15 to 60 minutes after the constant temperature amplification is carried out at the temperature of 60 to 65 ℃, and the method has the characteristics of simple operation, strong specificity, easy detection of products and the like. Upon DNA synthesis, pyrophosphate ions precipitated from deoxyribonucleic acid triphosphate substrates (dNTPs) reacted with magnesium ions in the reaction solution to generate a large amount of magnesium pyrophosphate precipitate, which appeared white. Therefore, the turbidity can be used as an index of the reaction, and whether amplification is carried out or not can be identified only by observing the white turbid precipitate with naked eyes without complicated electrophoresis and ultraviolet observation.
Because the loop-mediated isothermal amplification reaction does not need a PCR instrument and expensive reagents, the method has wide application prospect. Because the technology has high sensitivity, the problem of easy aerosol pollution caused by uncovering exists, most laboratories in China cannot be strictly partitioned at present, and the problem of false positive is serious. Therefore, the invention is improved in the aspect of reducing pollution, and a microfluidic system is integrated on a honeycomb chip, so that the high-throughput rapid detection is facilitated on one hand, and primers are anchored on the chip and provided with microfluidic capillaries on the other hand, so that the pollution is reduced. In the hydatid detection, the method still stays at the single-tube detection of the LAMP at present, and the preparation of a related gene chip aiming at intestinal protozoa by a solid phase carrier is not carried out. The invention can realize the identification of a plurality of insect species in the excrement sample by simultaneously and efficiently detecting.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide a gene chip kit for efficiently detecting two types of hydatid. The invention takes two common and seriously harmful echinococcus granulosus and echinococcus multilocularis of echinococcus as research objects, and establishes a rapid, sensitive and specific detection kit.
The second technical problem to be solved by the invention is to provide a microfluidic coupling LAMP detection method for echinococcus granulosus and echinococcus multilocularis. The synthesis of the specific primers of two kinds of cochleariae LAMP solves the tedious steps of carrying out multiple PCR detection and electrophoresis, one-time reaction can achieve the purpose of detecting multiple genes simultaneously, the types of the cochleariae are determined, the workload is greatly reduced, the detection time is shortened, the detection cost is reduced, and the development trend is larger.
In order to solve the technical problems, the invention is realized by the following technical scheme:
in a first aspect of the invention, there is provided a honeycomb chip-based method for detecting echinococcus (I), (II), (III)Echinococcosis) Rapid detection method for different species of insects for simultaneous detection of echinococcosis granulosa (Echinococcosis granulosus, E.granulosus) And echinococcosis multilocularis disease (A), (B), (C)Echinococcus multilocularis，E. multilocularis) 2 insect strains in total, wherein the kit comprises the following primers:
the echinococcus granulosus primer sequence is shown in SEQ ID NO. 1-4;
the echinococcus multilocularis primer sequence is shown in SEQ ID NO. 5-8.
As a preferred technical scheme of the invention, the kit also comprises a LAMP reaction system as follows:
as the preferable technical scheme of the invention, the primer is freeze-dried and then solidified on the chip to prepare the honeycomb chip; the kit further comprises a color-developing agent, for example, calcein or the like.
In a second aspect of the present invention, there is provided a method for detecting different species of echinococcus based on honeycomb chip, the method comprising the steps of:
(1) designing and screening a primer; the designed primer sequence is as follows: the echinococcus granulosus primer sequence shown in SEQ ID NO. 1-4; the echinococcus multilocularis primer sequence shown in SEQ ID NO. 5-8;
(2) plasmid construction: adopting gene synthesis as a template, and constructing a plasmid as verification; each insect strain has a respective template;
(3) establishing a single-tube LAMP method, and carrying out LAMP detection on the designed primers to obtain effective amplification primers;
(4) performing amplification specificity verification of the gene chip according to the LAMP reaction condition in the step (3);
(5) performing sensitivity verification on the gene chip according to the LAMP reaction conditions in the step (4);
(6) further performing in vitro encapsulation verification by using the primer;
(7) and detecting the actual sample by using the primer.
As a preferred technical scheme of the invention, the step (2) is specifically as follows:
the nucleotide sequence of the echinococcus granulosus template plasmid is shown in SEQ ID NO. 9-10;
the nucleotide sequence of the echinococcus granulosus template plasmid is shown in SEQ ID NO 11.
In a preferred embodiment of the present invention, in the step (3), the step (4) and the step (5), the LAMP reaction conditions are as follows:
after mixing the above, the PCR instrument was left at 60 ℃ for 1 hour, and then fluorescence was observed, and electrophoresis was performed.
In a third aspect of the invention, a primer set for detecting different species of echinococcus is provided, comprising the following primer sequences:
the echinococcus granulosus primer sequence is shown in SEQ ID NO. 1-4;
the echinococcus multilocularis primer sequence is shown in SEQ ID NO. 5-8.
In the fourth aspect of the invention, the application of the primer group in the preparation of the kit for detecting different echinococcus species based on the honeycomb chip is provided.
In the fifth aspect of the present invention, there is provided a method for detecting biological nucleic acid of different species of echinococcus, comprising the steps of:
(1) freeze-drying the primer and then solidifying the primer on a chip to prepare the LAMP microfluidic chip;
(2) selecting a proper LAMP reaction system, adding a color developing agent, and adding the mixture into a chip;
(3) the reaction result can be judged by naked eyes or instruments.
As a preferred technical scheme of the invention, the step (1) is specifically as follows: assembling a plurality of capillaries into a customized base for fixing to form the LAMP microfluidic chip; then, dissolving the primers in a primer fixing solution, respectively sucking the primer fixing solutions aiming at different targets, and adding the primer fixing solutions into corresponding capillary reaction holes on a capillary chip according to a pre-designed arrangement sequence; reserving two capillaries, wherein one capillary fixes a positive control primer group as a positive control hole, and the last capillary fixes a non-fixed primer group as a negative control hole; drying the capillary to fix the primer on the inner wall of the capillary.
As a preferred technical scheme of the invention, the step (2) is specifically that a sample to be detected, an LAMP reaction reagent and a color developing agent are added into a reaction hole of the microarray chip and reacted for 1 hour at 63 ℃.
As a preferred technical scheme of the invention, the reaction result detection and data analysis in the step (3) are specifically as follows: after the LAMP reaction is finished, the color development condition of all reaction holes on the chip is detected through ultraviolet fluorescence, and the detected result is judged to be positive or negative under the condition that the positive control hole develops color and the negative control hole does not develop color.
Compared with the prior art, the invention has the beneficial effects that:
in the method for detecting echinococcus type 2 tapeworm, the bioinformatics analysis is carried out on the two types of echinococcus tapeworms, primers capable of distinguishing the two types of echinococcus tapeworms simultaneously are designed, time cost can be greatly saved, and operation difficulty is reduced.
2 the primer of the invention has good specificity and can distinguish the targets of two types of echinococcus.
3 the primer of the invention has good sensitivity, and can simultaneously detect the mixed template with the concentration of 5 ng/. mu.l. Wherein the detection range of the primer of the A type can reach the concentration of 1 pg/mu l grade.
4 the honeycomb chip detection method of the invention can be used for quickly and efficiently detecting the target in the actual sample, and is beneficial to conveniently and quickly screening echinococcus infection samples.
The invention 5 is improved in the aspect of reducing pollution, and a microfluidic system is integrated on a honeycomb chip, so that the high-flux rapid detection is facilitated, and the pollution is reduced by anchoring a primer on the chip and having a microfluidic capillary.
6 in the hydatid detection, at present, two types of gene chips for detecting the hydatid by a single tube of LAMP and preparing the hydatid on a honeycomb chip are still remained, and the preparation of related gene chips for intestinal protozoa by a solid phase carrier is not carried out. The invention can realize the identification of a plurality of insect species in the excrement sample by simultaneously and efficiently detecting.
Various verifications show that the kit has the characteristics of rapidness, sensitivity and strong specificity, solves the problem that the basic layer does not have the complicated steps of PCR instrument detection and electrophoresis, can simultaneously detect a plurality of genes by one-time reaction, defines the insect species of the insect species, greatly lightens the workload, shortens the detection time, reduces the detection cost and has greater development tendency.
Drawings
FIG. 1 is a schematic diagram showing the LAMP amplification result of the primer screening of echinococcus granulosus (type A) in example 3 of the present invention. In FIG. 1, primers 1-10 are designed autonomously and tested, and 1-5 Echinococcus granulosus (type A) template is used as a detection sample, and test primers of 5 groups respectively are tested to find that the fourth group has typical positive amplification; 6-10 the echinococcus multilocularis (B type) template is used as a detection sample, and 5 groups of test primers are respectively tested, so that no typical positive amplification is found. On the right side, electrophoresis images 1-10 represent the same as above, and M represents a cotton template, which serves as a positive control for the single-tube detection reaction system.
FIG. 2 is a schematic diagram showing the LAMP amplification result of the primer screening of echinococcus multiplex (type B) in example 3 of the present invention. In FIG. 2N 1, P1 represents primers and primer plus template with positive control cotton MON 88913; n2, P2 represents E-gm primers and primer plus template; n3, P3 represents primers and primer plus template of Em-O; n4, P4 represent primers and primer plus template for Em-N.
FIG. 3 is a schematic diagram of the cross reaction (specificity) experiment between species detected by the gene chip in example 4 of the present invention.
FIG. 4 is a diagram showing the sensitivity test of the primers for Echinococcus amphitypus in the single-tube test in example 4 of the present invention. In FIG. 4M represents positive control cotton MON88913,1-5 represent type A primers at concentrations from 10 ng/. mu.l, 1 ng/. mu.l, 100 ng/. mu.l, 10 ng/. mu.l, 1 '-5' represent type B Em-N primers at concentrations from 10 ng/. mu.l, 1 ng/. mu.l, 100 ng/. mu.l, 10 ng/. mu.l, 1 ng/. mu.l.
FIG. 5 is a graph showing the results of sensitivity experiments of Echinococcus A and B primers in a honeycomb chip in example 5 of the present invention.
FIG. 6 is a graph showing in vitro encapsulation results of example 6 of the present invention.
FIG. 7 is a graph showing the results of detection of the test sample in example 7 of the present invention; in fig. 7: 1. positive control: cotton template MON 88913; 2. encapsulation Em; eg, 3-8.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Experiment platform
The detection platform on which the invention is based is a honeycomb chip (platform) provided by Shanghai university of transportation, and the specific content of the detection platform is disclosed in the patent application with the patent application number of 201510901558.1 and the invention name of the invention is a high-throughput rapid detection method of nucleic acid based on capillary microarray. The honeycomb chip (platform) is processed into a microarray by means of capillary assembly, casting, machining and the like, a plurality of hydrophilic vertical microchannels are contained in the microarray, and the outer surface of the microarray is subjected to super-hydrophobic modification by a chemical method; adding a plurality of groups of nucleic acid amplification primers into a plurality of micro-pipelines respectively, drying and fixing, and placing the microarray into a reaction tube; then, introducing nucleic acid amplification reaction components into each micro-pipeline at one time by a special sample adding device in a siphon mode, and putting the components into a temperature control device for amplification reaction; real-time detection and end point detection are respectively realized by continuously measuring in the reaction or measuring the fluorescent signal in the micro-pipeline for one time after the reaction; the amplification product may also be recovered for subsequent use. The platform has the advantages that the detection of a plurality of nucleic acid targets can be quickly and conveniently realized in one reaction, and the platform is widely applied to the fields of nucleic acid multiplex detection, field detection and the like.
The honeycomb chip (platform) of the invention can simply realize the rapid parallel sample introduction of nucleic acid amplification reagents and the rapid parallel operation of amplification reaction in a micro system. Compared with the existing method, the method has the specific advantages of high detection flux, simple experiment operation, less sample consumption, low detection cost, no need of expensive equipment and the like. Can be widely applied in the fields of infectious disease rapid diagnosis, entry and exit inspection and quarantine, transgenic crop product field detection, food water source microorganism field detection, crime field evidence identification, biological counterterrorism and the like.
The specific detection method of the honeycomb chip (platform) comprises the following steps:
(1) adding a plurality of groups of nucleic acid amplification primers into a plurality of microchannels on a capillary microarray respectively, drying to facilitate the nucleic acid amplification primers to be attached to the inner walls of the microchannels, and fixing the capillary microarray in a transparent reaction tube;
(2) adding a nucleic acid amplification reaction component containing sample nucleic acid into a micro-pipeline to form a nucleic acid amplification system, and sealing a reaction pipe orifice;
(3) placing the reaction tube with the nucleic acid amplification system under a temperature-controlled condition for amplification reaction;
(4) real-time detection is realized by measuring continuous fluorescence signals of the amplification reaction or end-point detection is realized by measuring one-time fluorescence signals after the amplification reaction is finished.
Wherein, a plurality of micro-pipelines in the capillary micro-array penetrate and are arranged on the substrate in an array mode, and a small part of the end part of each micro-pipeline is exposed out of the surface of the substrate; the upper surface of the substrate, the outer surface of the micro-pipeline exposed out of the substrate part and the inner surface of the bottom end of the micro-pipeline are hydrophobic surfaces; the hydrophobic surface may be achieved by applying a layer of hydrophobic coating to the respective surface. The substrate is made of plastic, glass, metal and other high polymer materials; wherein, the other high molecular materials are polydimethylsiloxane, polymethyl methacrylate, polytetrafluoroethylene, rubber and the like; the micro-pipeline is a hydrophilic capillary, the part of the micro-pipeline exposed out of the substrate and the bottom end surface of the micro-pipeline are hydrophobic, and the micro-pipeline is made of hydrophilic materials. The step (1) is adding, in particular, a nucleic acid amplification primer is dissolved in a cross-linking agent and then added into a micro-pipeline; the cross-linking agent is any one of the following three mixed solutions:
a. 0.1-1% by mass of acetic acid aqueous solution of chitosan, and the pH value is 4.5-6.0;
b. gelatin water solution with the mass percent of 0.1-1%;
c. 0.05-5% of polyethylene glycol aqueous solution by mass percent.
The reaction component added in the step (1) is specifically introduced into each micro-pipeline in an inverted siphon mode. The inverted siphon mode is as follows: the sample cell of the sample adding device filled with the nucleic acid amplification reaction component solution is inserted into the reaction tube downwards, the nucleic acid amplification reaction component is contacted with the top end of the inner wall of the hydrophilic micro-tube and then quickly filled in the hydrophilic tube under the action of siphon, and then the sample adding device is removed. The measurement in (4) can be performed by means of fluorescence detection equipment or photometric detection equipment, or can be performed by visually distinguishing color or brightness differences, namely, before each measurement, a light source with corresponding emission wavelength is required to irradiate the reactant in the microchannel, and then the measurement is performed. In the method, the temperature control device in (3) and the measurement in (4) can be integrated into an automatic device, and the automatic operation of the automatic device is controlled by a software program. The method can be realized in a single reaction tube, or can be realized in parallel in an integrated 8-tube, 96-well plate or 384-well plate.
The platform has the advantages that: (1) by utilizing the capillary microarray and the hydrophilic and hydrophobic characteristics, the reaction liquid can be quickly and conveniently added into a plurality of microchannels at one time by adopting a specially designed sample adding device, so that the flux and the detection efficiency of one-time detection are improved; (2) the micro-pipeline is used as the reaction cavity, so that the dosage of a reagent sample is greatly reduced, and the detection cost is reduced; (3) the method is suitable for the fields of high-flux rapid detection of various nucleic acids, such as rapid detection of infectious diseases, entry and exit inspection and quarantine, food safety and transgenic detection, criminal investigation identification and the like.
Example 1 detection primer design for echinococcus amphitypus
(1) According to bioinformatics analysis, different target genes are respectively adopted for target design.
(2) Designing and screening a primer; the primer sequences of echinococcus granulosus (type A) and echinococcus multilocularis (type B) are as follows:
type A primers:
primers for type B:
example 2 plasmid construction;
in view of the high template complexity of the experimental sample, gene synthesis is adopted as a template, and plasmids are constructed for verification.
Each insect strain has its own template.
Aiming at the A-type insect species:
aiming at the type B insect species:
example 3 establishment of Single-tube LAMP method for LAMP detection of designed primers to obtain effective amplification primers
The LAMP reaction system adopted in this example is as follows:
after mixing the above, the PCR instrument was left at 60 ℃ for 1 hour, and then fluorescence was observed, and electrophoresis was performed.
First, primer screening for type a:
the hydatid mitochondrial gene sequence region also contains a plurality of genes, including not only NADH1 and NADH5, but also COI genes. Now, specific primer design of echinococcus granulosus is performed for COI. Sequence alignment shows that the region is too strongly conserved, highly homologous sequence information exists in various insects, and specificity is not enough. Thus, primer 5 set a was designed and primer matched length templates of type a and type B were synthesized simultaneously.
And (3) detection strategy: detecting A type, adopting two templates of A (SEQ ID NO. 9) and B (SEQ ID NO. 10) to confirm whether the A type has specificity; if both the results are positive, defining the result as universal type; if only A template is positive, the primer is specific to A. The screening primers are specifically shown in table 1:
TABLE 1
As shown in FIG. 1, type A was detected using two templates, A (SEQ ID NO. 9) and B (SEQ ID NO. 10); type B was detected using the template for B (SEQ ID NO. 11). The results show that 1 group of specific primers A and B was screened, and the detection was effective using the plasmid-synthesized template, as shown in Table 2.
TABLE 2
Second, primer screening for type B
Mitochondrial sequences have specificity of parting genes in hydatid, and the identification of two insect species is found by looking back the literature aiming at NADH dehydrogenase outburt genes, so that the NADH sequences of the two insect species are compared to find that the two insect species are highly similar and can not be distinguished; designing an AB universal type identifying primer 1 group and a B type identifying primer 2 group, wherein one group is from the literature, and the other group is a self-designed primer.
And (3) detection strategy: detecting general type and B type at the same time, if both results are positive, then the type is B; if the universal type result is positive and B is negative, the type A is determined. The primers screened are shown in Table 3:
TABLE 3
As shown in fig. 2, N1, P1 in fig. 2 represent primers and primer plus template with positive control cotton MON 88913; n2, P2 represents E-gm primers and primer plus template; n3, P3 represents primers and primer plus template of Em-O; n4, P4 represent primers and primer plus template for Em-N. Effective detection primers for Echinococcus multilocularis were obtained by Table 3.
Example 4 verification of amplification specificity of Gene chip according to LAMP reaction conditions of example 3
(1) Extraction of Encapsulated DNA
Is completed by adopting a tissue DNA extraction kit of the Tiangen.
(2) The chip composition is shown in table 4:
TABLE 4
Note that: (1) detecting indexes Eg and Em by three holes respectively, and repeating as three technologies; (2) two wells of positive and negative controls were used, and both techniques were repeated.
(3) Validation of primer effectiveness by single-tube LAMP
The LAMP reaction system adopted is as follows:
after mixing the above, the PCR instrument was left at 60 ℃ for 1 hour, and then fluorescence was observed, and electrophoresis was performed.
Results are shown in FIG. 3, single template primer validity test (3 reactions); the templates Eg-COI-template (positive), Em-COI-template (negative) and Em-N-template (positive) are respectively used for reacting with the chip, and the effectiveness of the primer on the chip is detected.
Example 5 Gene chip sensitivity verification was performed according to the LAMP reaction conditions of example 4;
(1) extraction of Encapsulated DNA
Is completed by adopting a tissue DNA extraction kit of the Tiangen.
(2) Validation of primer effectiveness by single-tube LAMP
The LAMP reaction system adopted is as follows:
after mixing the above, the PCR instrument was left at 60 ℃ for 1 hour, and then fluorescence was observed, and electrophoresis was performed.
As a result: and (3) carrying out sensitivity detection by adopting different template concentrations. Sad1 is a positive control for this system. The sensitivity of the primer of type A can reach 1 pg/mu l, and the detection sensitivity of the primer of type B can also reach 5 ng/mu l.
In the single tube primer sensitivity test, the experimental sample concentrations were set up as follows:
the results of the reactions with different concentration gradients diluted by the synthetic plasmid template are shown in FIG. 4, wherein M represents positive control cotton MON88913,1-5 represents primer concentrations for type A from 10 ng/. mu.l, 1 ng/. mu.l, 100 ng/. mu.l, 10 ng/. mu.l, 1 ng/. mu.l, and 1 '-5' represents Em-N primer concentrations for type B from 10 ng/. mu.l, 1 ng/. mu.l, 100 ng/. mu.l, 10 ng/. mu.l, and 1 ng/. mu.l. The result shows that the detection sensitivity of the Eg (A type primer) can reach 1 pg/mu l, and the detection sensitivity of the Em (B type primer) only reaches 10 ng/mu l, which indicates that the primer sensitivities of the two types of insects to be detected designed in the invention are different. FIG. 5 is a graph showing the sensitivity test results of the A and B Echinococcus types primers in the bee nest chip in example 5 of the present invention, if the primers of the two types of insects are bound in the bee nest chip for detection. We can find that the positive plasmid templates of two echinococcus tapeworm species can be simultaneously detected under the concentration of 5ng/ul, which also shows that the use of the honeycomb chip can improve the detection sensitivity of Em to 10 pg/ul.
Example 6 laboratory validation was performed using primers of type A and type B.
The DNA of a sample obtained by ordinary PCR amplification and sequencing is detected by the detection primer disclosed by the invention, and the specific primer disclosed by the invention can detect the type of the encapsulated insect species as shown in figure 6.
The echinococcus is spread in the intermediate host body to form new echinococcus, so the echinococcus is obtained from the internal organs of the intermediate host on site, and the verification of A type and B type is carried out. Echinococcus comprised of cyst wall and cyst contents (hair growth cyst, primary bursa, cyst fluid, etc.), and was purchased from blood/cell/tissue genome DNA extraction kit (DP304) of Tiangen Biotechnology Ltd by tissue DNA extraction kit, and stored at-20 ℃ according to the kit instructions. PCR mix was purchased from Bio-Server.
The identification of pathogens infected in the actual samples was carried out according to the reaction system of Table 6 and the reaction program of Table 7 with the primers (Table 5) in Minoru Nakao et al (Mitochondrial genetic code in sources, Molecular and Biochemical Parasitology, 2000, 111: 415-424) and Zhongzhen et al (comparison of 3 PCR methods for detecting Echinococcus animalis, J.International medical parasitosis, 2011,38 (3): 148-152).
Table 5: half nested PCR primer verification actual sample
The half nested PCR reaction system is the same as in Table 6, and the reaction procedure is shown in Table 7.
Table 6: single-tube PCR amplification reaction system
Table 7: semi nested PCR cycle parameters
And (3) identifying a PCR product: 5. mu.l of the PCR product was electrophoretically analyzed on a 2% agarose gel (120V/40 min).
Note that: each sample was amplified at least 3 times by parallel PCR, each time with positive and negative controls. Sequencing the obtained positive detection samples to prove that the positive detection samples are echinococcus granulosus and echinococcus multilocularis.
Example 7 detection of actual samples was performed using primers of type A and type B.
The method comprises the following steps:
(1) manufacturing and processing an LAMP microfluidic chip: assembling 10 hydrophobized capillaries into a customized base, and fixing for later use;
(2) and (3) primer fixation: dissolving the primers in the embodiment 1 in a primer fixing solution, respectively sucking the primer fixing solutions aiming at different targets, and adding the primer fixing solutions into corresponding capillary reaction holes on a capillary chip according to a pre-designed arrangement sequence; two capillaries are reserved, wherein one capillary fixes a positive control primer group as a positive control hole, and the last capillary fixes a non-fixed primer group as a negative control hole. Drying the capillary tube to fix the primer on the inner wall of the capillary tube;
(3) LAMP reaction on a chip: adding a sample to be detected, an LAMP reaction reagent and calcein into a reaction hole of the microarray chip through an auxiliary sample adding device, and reacting for 1 hour at 63 ℃;
(4) and (3) result detection and data analysis: after the LAMP reaction is finished, the color development condition of all reaction holes on the chip is detected through ultraviolet fluorescence, and the detected result is judged to be positive or negative under the condition that the positive control hole develops color and the negative control hole does not develop color.
The DNA of the samples obtained by the conventional PCR amplification and sequencing was detected by the detection primers of the present invention, and as shown in FIG. 7, it was found that 3 samples containing Eg verified by the conventional PCR were 66.7% identical to those detected by the Eg primers of the honeycomb chip (2/3). Two duplicate wells were used for each sample, i.e., 3 and 6, 4 and 7, 5 and 8 were duplicate wells. Where the Eg samples in 3 and 6 did not match, the analysis may be due to degradation of the nucleic acids. We also need a large number of subsequent samples for validation.
Sequence listing
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Claims (12)
1. A kit for detecting different species of echinococcus based on honeycomb chips is characterized in that a microfluidic system is integrated on a honeycomb chip and is used for detecting echinococcus granulosus and echinococcus multicavium simultaneously, and the kit comprises the following primers:
the echinococcus granulosus primer sequence is shown in SEQ ID NO. 1-4;
the echinococcus multilocularis primer sequence is shown in SEQ ID NO. 5-8.
2. The kit of claim 1, further comprising a LAMP reaction system as follows:
。
3. The kit of claim 1, wherein the primers are lyophilized and cured onto the chip to form a honeycomb chip; the kit also comprises a color developing agent.
4. A method for detecting different species of echinococcus based on honeycomb chips is characterized by comprising the following steps:
(1) designing and screening a primer; the designed primer sequence is as follows: echinococcus granulosus primer sequences set forth in SEQ ID No.1-4 according to claim 1; the echinococcus multilocularis primer sequence of SEQ ID No.5-8 of claim 1;
(2) plasmid construction: adopting gene synthesis as a template, and constructing a plasmid as verification; each insect strain has a respective template;
(3) establishing a single-tube LAMP method, and carrying out LAMP detection on the designed primers to obtain effective amplification primers;
(4) performing amplification specificity verification of the gene chip according to the LAMP reaction condition in the step (3);
(5) performing sensitivity verification on the gene chip according to the LAMP reaction conditions in the step (4);
(6) further performing in vitro encapsulation verification by using the primer;
(7) and detecting the actual sample by using the primer.
5. The method according to claim 4, wherein the step (2) is embodied as:
the nucleotide sequence of the echinococcus granulosus template plasmid is shown in SEQ ID NO. 9-10;
the nucleotide sequence of the echinococcus granulosus template plasmid is shown in SEQ ID NO 11.
6. The method of claim 4, wherein in step (3), step (4) and step (5), the LAMP reaction conditions are as follows:
After mixing the above, the PCR instrument was left at 60 ℃ for 1 hour, and then fluorescence was observed, and electrophoresis was performed.
7. The primer group for detecting different echinococcus species is characterized by comprising the following primer sequences:
the echinococcus granulosus primer sequence is shown in SEQ ID NO. 1-4;
the echinococcus multilocularis primer sequence is shown in SEQ ID NO. 5-8.
8. Use of the primer set according to claim 7 for the preparation of a honeycomb chip-based kit for the detection of different species of echinococcus.
9. A biological nucleic acid detection method for different echinococcus species is characterized by comprising the following steps:
(1) freeze-drying the primer of claim 7, and then solidifying the primer on a chip to prepare an LAMP microfluidic chip;
(2) selecting a proper LAMP reaction system, adding a color developing agent, and adding the mixture into a chip;
(3) the reaction result can be judged by naked eyes or instruments.
10. The method according to claim 9, wherein step (1) is specifically: assembling a plurality of capillaries into a customized base for fixing to form the LAMP microfluidic chip; then, dissolving the primers of claim 7 in the primer fixative, respectively sucking the primer fixative for different targets, and adding the primer fixative to corresponding capillary reaction holes on the capillary chip according to a pre-designed arrangement sequence; reserving two capillaries, wherein one capillary fixes a positive control primer group as a positive control hole, and the last capillary fixes a non-fixed primer group as a negative control hole; drying the capillary to fix the primer on the inner wall of the capillary.
11. The method according to claim 9, wherein the step (2) is carried out by adding the sample to be detected, the LAMP reaction reagent and the color-developing agent into the reaction well of the microarray chip and reacting at 63 ℃ for 1 hour.
12. The method of claim 9, wherein the reaction result detection and data analysis in step (3) are specifically: after the LAMP reaction is finished, the color development condition of all reaction holes on the chip is detected through ultraviolet fluorescence, and the detected result is judged to be positive or negative under the condition that the positive control hole develops color and the negative control hole does not develop color.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115961057A (en) * | 2022-12-29 | 2023-04-14 | 北京亿森宝生物科技有限公司 | Echinococcosis detection primer, probe, kit and application |
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| CN107365862A (en) * | 2017-08-29 | 2017-11-21 | 中国疾病预防控制中心寄生虫病预防控制所 | For detecting the primer and probe and its kit of Echinococcus granulosus in dog excrement |
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| CN106854674A (en) * | 2015-12-08 | 2017-06-16 | 上海交通大学 | A kind of nucleic acid high flux method for quick based on capillary microarray |
| CN107365862A (en) * | 2017-08-29 | 2017-11-21 | 中国疾病预防控制中心寄生虫病预防控制所 | For detecting the primer and probe and its kit of Echinococcus granulosus in dog excrement |
| CN108796091A (en) * | 2018-06-04 | 2018-11-13 | 青海大学 | Loop-mediated isothermal amplification is applied to the genotyping detection method and detection kit of more room/Echinococcus Granulosus Cysts |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115961057A (en) * | 2022-12-29 | 2023-04-14 | 北京亿森宝生物科技有限公司 | Echinococcosis detection primer, probe, kit and application |
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