CN109762910B - Primer and kit for simultaneously detecting two types of echinococcosis - Google Patents

Abstract

The invention discloses a primer and a kit for simultaneously detecting two types of echinococcosis, wherein the primer is arranged in the kit in a form of freeze-dried powder or dissolved in a buffer solution. The primers comprise three specific primer pairs, namely: echinococcus granulosus specific primer pairs, echinococcus canadensis specific primer pairs and Echinococcus multilocularis specific primer pairs. By using the three pairs of primers in the kit, single or mixed infection of three pathogens can be detected through one round of PRC amplification, and whether the detected sample has infection and insect species infection can be determined according to specific bands in the electrophoresis of amplified products. The kit provided by the invention is rapid and convenient to operate, and can accurately diagnose and identify echinococcosis caused by three echinococcosis infections.

Description

Primer and kit for simultaneously detecting two types of echinococcosis

Technical Field

The invention relates to a primer and a kit, in particular to a primer and a kit for simultaneously detecting two types of echinococcosis.

Background

Echinococcosis is a serious zoonotic infectious disease caused by infection of a plurality of insect species in echinococci, which seriously damages human health and causes huge social and economic burden. Echinococcosis is mainly divided into two types, namely, cystic disease and bubble disease, the former is extremely wide in distribution range, the disease burden of global patients reaches 1,009,662 thousands of DALYs (disability adjustment life years), and the direct economic loss of China exceeds 30 hundred million yuan each year; the latter is also called 'insect cancer', which can lead to liver cancer-like lesions with extremely high mortality rate, and the death rate of ten years of untreated mortality rate reaches 94%. China is one of the most serious world-wide echinococcosis epidemic countries, the number of the existing disease estimated people reaches 11.5 ten thousand, the number of sick livestock exceeds 5000 ten thousand per year, and the economic loss of people and livestock accounts for about 40% of the world, and the world is first. Echinococcosis is widely distributed in the northwest minority nationality area of China, and relates to 350 epidemic counties of 9 provinces except Tibet, and the threatened population exceeds 5000 thousands, which is an important cause of disease-induced and disease-induced return of people in the epidemic area.

Prevention and treatment of important parasitic diseases such as echinococcosis are one of important links for guaranteeing physical health of people and comprehensively promoting healthy Chinese construction. Echinococcus is a pathogen of human and livestock echinococcosis, and consists of 9 species, 4 of which are popular in China to different degrees, but only 3 of them, namely echinococcus pinnata (Echinococcus granulosus sensu strict) and echinococcus canadensis (Echinococcus canadensis), which cause cystic echinococcosis, and echinococcus multilocusta (Echinococcus multilocularis) which cause follicular echinococcosis are main subjects of current control and research of echinococcosis in China. Because the two types of echinococcosis has obvious different treatment principles and drug curative effects and obvious differences in the life history and transmission characteristics of three insect species, the accurate diagnosis and identification of the two types of echinococcosis are very important as the basis of effective treatment, scientific prevention and control of the echinococcosis.

At present, the diagnosis of the echinococcosis mainly depends on the image technology such as B ultrasonic and CT and pathological examination, and is easy to cause misdiagnosis, missed diagnosis and typing difficulties of small focus and atypical focus due to the limitation of the method, especially for the remote ethnic area where the echinococcosis is mainly popular, the problems of misdiagnosis and the like are more easy to occur due to the low general level of the diagnosis technology; for the low epidemic or non-epidemic area of echinococcosis, there is a lack of experience due to fewer contact cases, even in some trimethyl hospitals, there are also misdiagnosis or typing situations. Serological detection is another project, more echinococcosis diagnosis methods are adopted, but the existing detection kit is high in false positive and serious in cross contamination, and the detection result can only be used as an auxiliary basis for reference. At present, aiming at the diagnosis method of the echinococcosis, besides the defect of insufficient accuracy and difficulty in ensuring timeliness and effect of treatment, more importantly, the existing inspection means for the echinococcosis cannot realize the identification of the level of the insect species, in other words, the overgrowth condition of the insect species transmission in the environment cannot be judged according to the change of the infected insect species of a patient, so that the prevention and control strategy can be adjusted in time. For the above reasons, research and establishment of a detection method which is accurate, can realize result standardization judgment and can perform inter-species identification are urgently needed to meet the working requirements of echinococcosis prevention and treatment.

The PCR technology is increasingly applied to diagnosis and detection of various parasitic diseases due to the characteristics of rapidness, accuracy, sensitivity and the like, and has better application effects, such as malaria, toxoplasmosis and the like, a PCR detection method is established, and the PCR technology is widely popularized and applied in prevention and treatment work, so that the PCR technology plays an increasingly important role in the field of diagnosis of the parasitic diseases. As the only method capable of identifying the echinococcosis infected insect species at present, the PCR technology can realize the infection detection of human bodies and various intermediate and final animal hosts, and is the development direction and research hot spot of the current echinococcosis diagnosis and identification. However, the existing PCR detection method for echinococcus mainly uses single-tube amplification, and a pair of primers can only detect a corresponding echinococcus through single reaction, or sequencing identification is adopted, so that for the region where a plurality of insect species are mixed and popular, the method is complex in process, consumes time and consumes materials, and cannot meet the requirements of rapid diagnosis and large-scale investigation. According to public reports and data, no multiplex PCR primer pair for simultaneously detecting the echinococcus granulosus, the echinococcus multiloculas and the echinococcus canadensis exists at present, and only a multiplex PCR amplification method aiming at other insect species is found, so that the method is not suitable for the control work of the current echinococcus epidemic situation in China.

Disclosure of Invention

Aiming at the prior art, the invention provides a primer for simultaneously detecting two types of echinococcosis and a kit containing the primer, so as to achieve the purposes of rapidly and accurately diagnosing and identifying the echinococcosis.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: there is provided a primer for simultaneous detection of echinococcosis of two types, the primer comprising three specific primer pairs, wherein:

primer pair 1: the sequences are shown in SEQ ID NO.1 and SEQ ID NO.2;

primer pair 2: the sequences are shown in SEQ ID NO.3 and SEQ ID NO.4;

primer pair 3: the sequences are shown in SEQ ID NO.5 and SEQ ID NO.6.

The invention also constructs a kit based on the primer, and the obtained primer exists in the kit in the form of freeze-dried powder or dissolved in a buffer solution.

In addition to the primer, the kit also comprises a 2 xTaq PCR amplification reagent containing Taq DNA polymerase, dNTPs, mgCl 2, a reaction buffer solution, gel sample-loading dye and nuclease-free water.

The beneficial effects of the invention are as follows:

1. by using the primer and the kit provided by the invention, all three kinds of echinococcus species which are popular in China and have infectivity to human bodies can be detected and identified, and meanwhile, other non-target organisms or areas can not be amplified.

2. The primer and the kit provided by the invention can detect all 3 types of echinococcus canadensis which are found to be popular in China and have pathogenicity on human bodies at present through single-tube multiplex PCR reaction, and can be used for identifying single and mixed infections at the same time, so that the experimental time is effectively shortened, and the method is efficient, simple, convenient, economic and quick to operate, and is suitable for quick diagnosis and accurate prevention and control of echinococcosis under the current situation of echinococcus canadensis epidemic in China.

Drawings

FIG. 1 is an electrophoretogram of PCR results of 3 pairs of specific detection primers for specific amplification of three target Echinococcus single or mixed DNA templates, respectively;

FIG. 2 is an electrophoretogram of the PCR result of 3 pairs of specific detection primers for respectively detecting DNA templates of three kinds of echinococcus of interest and seven kinds of non-target insects with relatively close relativity;

FIG. 3 is an electrophoretogram of the PCR result of the sensitivity detection of 3 pairs of primers on three Echinococcus DNA templates of different concentrations respectively;

FIG. 4 is an electrophoretogram of PCR results of three repeated experiments of 3 primer pairs at different times respectively using human, livestock and small mammal focus samples as DNA templates.

Detailed Description

The invention aims to realize the purpose of diagnosing and identifying two types of echinococcosis caused by single-tube PCR amplification or mixed infection of three echinococci species, and develops a kit which comprises three specific primers, wherein the kit can detect narrow-sense echinococci granulosa, canadian echinococcus and echinococcus multiloculas simultaneously through single-tube amplification, and can remarkably improve the diagnosis accuracy and efficiency of the echinococcosis.

The following describes the present invention in detail with reference to examples.

Example one primer design

The published mitochondrial gene reference sequences of echinococcus narrowly defined on GenBank (NC_ 008075.1), echinococcus canadensis (NC_ 011121.1, AB235848.1, MG 597240.1) and echinococcus multilocusts (NC_ 000928.2) were searched for, and specific primers for the above 3 insect species genes were designed and the Primer mass was evaluated by combining Primer Premier 6.0 and Oligo 7 software with manpower. Through multiple rounds of screening, the 3 pairs of primers can be finally determined to be capable of rapidly, accurately and simultaneously detecting and identifying 3 echinococcus, and the sequences of the primers are as follows:

(1) Echinococcus granulosus in narrow sense

Upstream primer F1:5'-GTCTGTGTTTCTTACCATTG-3' (SEQ ID NO. 1)

Downstream primer R1:5'-GACCCGTACAAACATATATCAAC-3' (SEQ ID NO. 2)

(2) Echinococcus multilocularis

The upstream primer F2:5'-TTGTTCTTTGTGTTACTGTAGG-3' (SEQ ID NO. 3)

Downstream primer R2:5'-CTATACAGACATTGATTACCATAA-3' (SEQ ID NO. 4)

(3) Echinococcus Canadian tapeworm

Upstream primer F3:5'-GTAAGTCTAAGTTGGTTATTATTCAC-3' (SEQ ID NO. 5)

Downstream primer R3:5'-CTTATTAAACAACACAAAAATACTAAATG-3' (SEQ ID NO. 6)

Example two establishment of multiplex PRC amplification method

1. Extraction of genomic DNA from a sample to be tested

15-25 mg of insect body tissue or infection focus of human body and animal is taken, and the whole genome DNA of the sample is extracted according to the instruction book of the tissue DNA extraction kit.

2. Amplification system

Using a 25. Mu.l reaction system, 12.5. Mu.l of 2 XTaq PCR amplification reagent, 0.5. Mu.l of each of the upstream and downstream primers of the 10. Mu.M F1/R1 and F2/R2 primer pair, 1.5. Mu.l of each of the upstream and downstream primers of the 10. Mu.M F3/R3 primer pair, 6.5. Mu.l of nuclease-free water and 1. Mu.l of sample template DNA were added. The system was prepared as described above using nuclease-free water as a template, as a negative control.

3. Reaction conditions

The prepared PCR reaction tube is put into a PCR instrument, and the reaction procedure is set according to the following conditions: pre-denaturation at 94℃for 2min; denaturation at 94℃for 30s, annealing at 55℃for 45s, elongation at 72℃for 1min,40 cycles; lengthening and extending for 5min at 72 ℃.

Example three identification of PCR reaction products

Taking 10 mu l of PCR amplified products, carrying out electrophoresis on the products in 2% (w/V) agarose gel at a constant pressure of 3-5V/cm for 30-60 min, observing the results in a gel imaging analysis system, and judging whether the infections and the echinococcus species are present or not according to the presence and the size of the target bands by taking the DNA molecular weight standard as a reference.

If the length of the amplified product band is 811bp, the amplified product band is judged to be positive for infection of the echinococcus narrowly-defined, if the amplified product band is 315bp, the amplified product band is judged to be positive for infection of the echinococcus canadensis, and if the amplified product band is 457bp, the amplified product band is judged to be positive for infection of the echinococcus multinomus; if the amplified products are simultaneously provided with more than two or three length strips, judging that the mixed infection of the corresponding two or three echinococcus is positive; if the amplified product is not banded, it is judged as negative. If the negative control showed an amplified band, the experiment was not effective.

Example four specificity determination

The three primer pairs and the multiplex PCR amplification method provided by the invention are adopted, the multiplex PCR amplification is carried out by adopting the following three modes according to the operations in the second to third embodiments, and the specificity of the detection method is as follows:

(1) Respectively carrying out PCR amplification by taking DNA of the echinococcus multilocularis as a template;

(2) Selecting at least two of fine particles, canada and Echinococcus multilocularis, and performing PCR amplification by taking DNA of the at least two species as a template;

(3) Seven kinds of parasites which are popular in China and have relatively close relatives to the fine-grained, canadian and echinococcus multilocularis are selected, wherein the parasites comprise echinococcus crassifolium, taenia solium, taenia suis, taenia asiatica, taenia jaggy, taenia meganeck and Taenia longum, and DNA (deoxyribonucleic acid) of the parasites is used as a template for PCR (polymerase chain reaction) amplification.

The amplification results are shown in FIGS. 1 and 2. FIG. 1 is an electrophoresis chart of PCR results of specific amplification of 3 pairs of specific detection primers provided by the invention on three kinds of target echinococcus single or mixed DNA templates, respectively, wherein: lanes 1 to 3 are PCR amplification results of DNA templates of the echinococcus granulosus, the echinococcus multinomus and the echinococcus canadensis respectively, lanes 4 to 6 are PCR amplification results of mixed DNA of the echinococcus granulosus, the echinococcus granulosus and the echinococcus canadensis, the echinococcus multinomus and the echinococcus canadensis respectively, lanes 7 are PCR amplification results of mixed DNA of the echinococcus granulosus, the echinococcus canadensis and the echinococcus canadensis, lanes 8 are negative control, and M is a DNA molecular weight standard Marker II. FIG. 2 is an electrophoresis chart of PCR results of specific detection primers for three kinds of target echinococcus and seven kinds of non-target echinococcus DNA templates with relatively close relativity, wherein lanes 1-3 are PCR amplification results of the fine-grained, multi-chamber and Canadian echinococcus DNA templates respectively, lanes 4-10 are PCR results of the echinococcus strainodactylus, echinococcus suis, echinococcus and Echinococcus, 11 are negative controls, and M is DNA molecular weight standard Marker II. As can be seen from the figures: three pairs of specific primer pairs in the kit can simultaneously and specifically detect the fine particles, canadian and echinococcus multilocularis, the Canadian and echinococcus multilocularis and mixed infection of the fine particles, canadian and echinococcus multilocularis, and only target strips corresponding to insect species are amplified, in addition, the three pairs of primer pairs do not react with other 7 non-target parasites, and no strips are generated; the three pairs of primers provided by the invention can detect three corresponding target echinococcus, do not have cross reaction with each other, do not generate nonspecific reaction with other common parasites with relatively close relativity, and have relatively good specificity.

Example five determination of PCR amplification sensitivity

The original template DNA concentration was measured with a micro ultraviolet spectrophotometer using three kinds of target Echinococcus DNA as templates, and diluted with DNA extraction kit eluent to give final template concentrations of 0.4 ng/. Mu.l, 0.2 ng/. Mu.l, 0.04 ng/. Mu.l, 0.036 ng/. Mu.l, 0.032 ng/. Mu.l, 0.028 ng/. Mu.l, 0.024 ng/. Mu.l, 0.02 ng/. Mu.l, 0.016 ng/. Mu.l, 0.012 ng/. Mu.l, 0.008 ng/. Mu.l, 0.004 ng/. Mu.l in the PCR system, and multiplex PCR amplification was performed according to the procedures in examples two to three, to detect the method sensitivity.

The electrophoresis results are shown in FIG. 3, wherein FIG. 3 shows the electrophoresis graphs of the PCR results of the 3 pairs of primers provided by the invention for respectively carrying out sensitivity detection on three kinds of echinococcus DNA templates with different concentrations, wherein A is the PCR amplification result of echinococcus narrowly defined, B is the PCR amplification result of echinococcus multiloculas DNA, C is the PCR amplification result of echinococcus canadensis DNA, the final concentration of the three kinds of echinococcus DNA templates is 0.4ng/μl, 0.2ng/μl, 0.04ng/μl, 0.036ng/μl, 0.032ng/μl, 0.028ng/μl, 0.024ng/μl, 0.016/μl, 0.012ng/μl, 0.004/μl,13 lanes are negative controls, and M is DNA molecular weight standard Marker II. It can be seen that the detection limit of the three target insect species DNA provided by the invention can reach 0.004 ng/. Mu.l, which shows that the invention has higher sensitivity.

Example six primer application and reproducibility comparison

4 patients, 3 animals and 3 small mammal lesions were collected, DNA extracted, and the infected insect species were identified by sequencing. Using 10 of these specifically identified DNAs as templates, multiplex PCR amplification was performed according to the procedure of examples two to three, and repeated three times at different times.

The electrophoresis results are shown in FIG. 4, wherein FIGS. 4A-C are respectively electrophoresis graphs of PCR results using 10 focus samples of the same patient, livestock and small mammal as DNA templates at different times; lanes 1, 2, 5, 7 and 8 in the electrophoresis chart are PCR amplification results of the echinococcus granulosus DNA template, lanes 3, 6, 9 and 10 are PCR amplification results of the echinococcus multinomus DNA template, lane 4 is detected as the PCR amplification result of the echinococcus canadensis DNA template, lane 11 is a negative control, and M is a DNA molecular weight standard Marker II. As can be seen from the figure, the amplification result of the primer pair provided by the invention is completely consistent with the sequencing result, which shows that the primer pair can be effectively applied to detection of clinical and field samples. The results of the three repeated detection carried out at different times are consistent, which shows that the repeatability is good.

Example seven construction kit

The kit body is formed by a plurality of partitions so as to accommodate and fix a plurality of containers such as tubes or vials, wherein the containers respectively contain primers, and the 2 xTaq PCR amplification reagent containing Taq DNA polymerase, dNTPs, mgCl 2, reaction buffer, gel sample-loading fuel, nuclease-free water and the like. The primer is contained in a container in the form of freeze-dried powder, or the primer pair is dissolved in TE buffer solution and then contained in the container. By adopting the kit, PCR amplification reaction can be carried out by only adding a detection sample, and the reaction product can be directly loaded for gel electrophoresis analysis.

Although specific embodiments of the invention have been described in detail with reference to the examples and the accompanying drawings, it should not be construed as limiting the scope of protection of this patent. Various modifications and variations which may be made by those skilled in the art without the creative effort are within the scope of the patent described in the claims.

Sequence listing

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Claims (1)

1. The application of the primer for rapidly detecting the two-type echinococcosis in preparing a kit is characterized in that the primer for rapidly detecting the two-type echinococcosis consists of a echinococcus granulosus detection primer, a echinococcus multilocusts detection primer and a echinococcus canadensis detection primer; the narrow echinococcus granulosus detection primer consists of a sequence shown in SEQ ID NO:1-2 and a downstream detection primer; the echinococcus multilocularis detection primer consists of a sequence shown in SEQ ID NO:3-4, and a downstream detection primer; the Echinococcus canadensis detection primer consists of a sequence shown in SEQ ID NO:5-6, and a downstream detection primer; the kit is used for detecting narrow echinococcus granulosus, echinococcus multinomus and echinococcus canadensis.

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