CN107245521B - Primer and probe sequence for LAMP-LFD detection of heterodera simplicissima/heterodera pekinensis - Google Patents
Primer and probe sequence for LAMP-LFD detection of heterodera simplicissima/heterodera pekinensis Download PDFInfo
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Abstract
The invention discloses a primer and a probe sequence for LAMP-LFD detection of aphelenchoides simplex/aphelenchoides pekinensis, which are characterized by comprising three pairs of LAMP primers aniITS-F3 and EtaompA-B3; AniITS-FIP, AniITS-BIP; AniITS-LF, AniITS-LB; and a probe AniITS-HP, the nucleotide sequence is shown as SEQ NO1-NO7, the primer and the probe are utilized, the LAMP reaction system amplification step and the probe hybridization LAMP reaction product step are carried out through the LAMP reaction system, and the LFD detection step is used for realizing the visual detection of the simple heterodera cuneata/heterodera peichiana, so that the method has the advantages of high detection speed, time and labor saving, small dependence on instruments and equipment, and is more suitable for detection of basic-level mechanisms and field epidemic sources.
Description
Technical Field
The invention relates to a primer and a probe sequence for detecting heterodera cuneata, in particular to a primer and a probe sequence for detecting LAMP-LFD of simple heterodera cuneata/heterodera pethidius.
Background
Heterodera sinensis (A), (B) and (C)Anisakisspp.) are commonly parasitic in the gut and muscle tissues of teleost fish, causing allergic symptoms when humans eat raw, semi-finished or cured fish, resulting in anisakiasis. With the popularization of edible fresh fish fillets, the incidence rate of the anisakid nematode disease is increased year by year. In 2013, the famous book of quarantine and epidemic diseases of animals in the republic of China classifies the heterodera sinensis as a second type of infectious disease, and the infectious disease limits the import and export trade of marine products to a certain extent (http:// www.aqsiq.gov.cn). Infection with anisakidia disease mainly associated with simple anisakidia: (A. simplex) And (4) correlating. The study shows that there are three species of heterodera sinensis, i.e. simple heterodera sinensis: (A. simplexs. str.), anisakis peterii: (A. pegreffii) And C type heterodera cuneata: (A. simplexC) Three types of the nematode feed are very widely distributed, and researches show that the infection rate of the heterodera simplicissima and the heterodera pekinensis in fishes and cephalopods reaches 80 percent. Moreover, the two nematodes are parasitic in china relatively widely. Therefore, the establishment of a new method for quickly, accurately and sensitively detecting the simple heterodera cuneata/heterodera pekinensis has important significance for early diagnosis, early warning and real-time monitoring of diseases.
Currently, species of anisakis are identified primarily by traditional morphological observations. However, this method requires a long detection period and requires a professional to perform the detection. In recent years, many scholars at home and abroad have established molecular identification means based on PCR: SSCP method, conventional PCR, multiplex PCR, PCR-RFLP and sequencing of ribosomal and mitochondrial DNA. The methods for these molecules require complex instrumentation and skilled personnel.
Notomi reports that loop-mediated isothermal amplification (LAMP) can complete detection more rapidly and conveniently under isothermal conditions, and the technology attracts attention in various fields. LAMP products can be detected by means of ladder-like bands visualized by Agarose Gel Electrophoresis (AGE), turbidimetry with naked eyes, nucleic acid staining (SYBR Green I, calcein, etc.), and the like. Real-time fluorescence-based monitoring also allows for easy and intuitive detection, but real-time fluorescence instruments are expensive. The lateral flow test strip (LFD) is a novel method, and can detect LAMP products efficiently and accurately. Specifically hybridizing a biotin-labeled LAMP product with a Fluorescein Isothiocyanate (FITC) -labeled probe, and combining the hybridization product with an anti-FITC antibody on an LFD to form an immune complex. When the detection line passes through the LFD, the biotin ligand is captured and is combined with the detection line; the uncaptured will pass through the detection line and bind to the quality control line. The method is originally applied to the detection of aquatic pathogens, viruses and bacteria, and is also applied to veterinary medicine and plant disease detection nowadays. At present, the application of the technology to the detection of the simple heterodera cruda/heterodera pekinensis is not reported at home and abroad.
Disclosure of Invention
The invention aims to solve the technical problem of providing a primer and a probe sequence for LAMP-LFD detection of the simple heterodera cuneata/heterodera pekinensis, which have high detection speed, low detection cost and high detection sensitivity and accuracy.
The technical scheme adopted by the invention for solving the technical problems is as follows: a primer and a probe sequence for LAMP-LFD detection of heterodera simplicissima/heterodera pekinensis are disclosed, wherein three pairs of primer sequences and one probe sequence of LAMP are designed according to rDNA-ITS2 segment of the heterodera simplicissima/heterodera pekinensis (GenBank accession number: KU 991876), and the primer sequences are specifically as follows:
AniITS-F3：5’- GAATTGCAGACACATTGAGC -3’
AniITS-B3：5’- ATAGTAGATTCGGTGTTGACG -3’，
AniITS-FIP：5’- GTAATTCGACCCTCAGCCAGACACTAAGAATTCGAACGCACA -3’，
AniITS-BIP：5’- CTGTGAAGCATTCGGCAAGCAACCGCTCGTCATATTGTC -3’，
AniITS-LF：5’- TGCCATCGGGAATGAACC -3’，
AniITS-LB：5’- TTGCTGTTGTGTTGTTGGTG -3’，
probe AniITS-HP: 5'-GGTGAACTGTCTTCACGGTT-3' the flow of the air in the air conditioner,
wherein, the 5' end of AniITS-FIP is a biotin label; the 5' end of the probe AniITS-HP is labeled by fluorescein isothiocyanate.
The specific detection steps for detecting the heterodera simplicissima/heterodera pekinensis are as follows:
1) configuring an LAMP reaction system: the final concentration of each component of the reaction system is as follows: 0.2 mu mol/L of each of outer primers aniITS-F3 and aniITS-B3, 1.6 mu mol/L of each of inner primers aniITS-FIP and aniITS-BIP, 0.4 mu mol/L of each of loop primers aniITS-LF and aniITS-LB, 1.4 mmol/L of dNTPs, Tris-HCl 20 mmol/L of pH 8.8, KCl 10mmol/L, MgSO46.5mmol/L，(NH4)2SO410mmol/L, Triton X-1000.1%, 8U Bst DNA polymerase large fragment (New England Biolabs) and 2 muL sample template, and adding double distilled water to make the total volume of the reaction system be25 µl；
2) Amplification of an LAMP reaction system: performing amplification reaction on the reaction system, wherein the amplification reaction temperature is 63 ℃, and the amplification reaction time is 40 min;
4) probe hybridization and LFD detection: adding 20 pmol of probe AniITS-HP into the reaction system after amplification, incubating for 5 min at 63 ℃, hybridizing, adding 5 muL of hybridization solution into 100 muL of buffer solution, mixing uniformly, then immersing the LFD test strip into the buffer solution added with the hybridization solution for color development, and judging the result of LAMP amplification detected by the lateral flow test strip.
Compared with the prior art, the invention has the advantages that:
1. the detection sensitivity is high, the method is based on single heterodera simplex/heterodera pekinensis L3, and the detection sensitivity is 10 of monomer L3 polypide genome-5And (4) doubling.
2. The detection time is short, the amplification reaction only needs 40 min, the whole detection can be finished within 50min from the extraction of the sample genome DNA to the judgment of the finished result, the detection time is shortened by more than 1.5 h compared with the conventional PCR detection technology, and the detection speed is greatly improved.
3. The specificity is strong, the used specific primers are designed according to eight different regions of rDNA-ITS2 sections of the heterodera simplicissima/heterodera pekinensis, and the specific probe of DNA is also arranged, so that the false positive problem caused by methods such as agarose gel electrophoresis, fluorescent dye and the like can be effectively avoided.
4. The requirements of instruments and equipment are low, a PCR instrument, a gel electrophoresis system, an imaging system and the like used in the conventional PCR are not needed, and the detection can be finished only by one water bath.
5. The operation is simple, the result is obvious, the whole detection process does not relate to complex instruments and equipment, and people with little molecular biology basis can complete the operation; the detection result is clear and obvious, and can be judged by visual observation.
In conclusion, the primers and the probes of the invention are used for detecting the simple heterodera cuneata/heterodera pekinensis by the LAMP-LFD method, have higher rapidity, specificity and sensitivity, have simple instrument requirements, and can meet the requirements of basic level detection mechanisms and field epidemic source detection.
Drawings
FIG. 1 shows the result of LAMP specificity experiment of Heterodera simplex/Heterodera pekinensis; m: 100 bp Plus DNAsadeder (Fermentas, USA); NC: double distilled water is used as a template; asi: using the genome DNA of the aphelenchoides simply as a template, Ape: using the genomic DNA of the heterodera pekinensis as a template, and adding Aph, Aty, Con, Cmu, Had, Hfa, Rlo, Pde, Aca, Nme, Alu, Asu, Hco and Cel: respectively taking the genome DNA of the sperm whale heterodera, the typical heterodera, the caecum croucheri, the gray sea eel towards the caecum, the introverted uterine grease nematode, the fistulae iuria, the ascaris lumbricoides, the pseudodinavian neonematode, the echinocandia, the merebriella meretrica, the ascaris lumbricoides, the ascaris suum, the haemonchus contortus and the caenorhabditis elegans as templates.
FIG. 2 shows the result of LAMP-LFD specificity test of Heterodera simplex/Heterodera pekinensis; NC: double distilled water is used as a template; asi: using the genome DNA of the aphelenchoides simply as a template, Ape: using the genomic DNA of the heterodera pekinensis as a template, and adding Aph, Aty, Con, Cmu, Had, Hfa, Rlo, Pde, Aca, Nme, Alu, Asu, Hco and Cel: respectively taking the genome DNA of the sperm whale heterodera, the typical heterodera, the caecum croucheri, the gray sea eel towards the caecum, the introverted uterine grease nematode, the fistulae iuria, the ascaris lumbricoides, the pseudodinavian neonematode, the echinocandia, the merebriella meretrica, the ascaris lumbricoides, the ascaris suum, the haemonchus contortus and the caenorhabditis elegans as templates.
FIG. 3 shows the result of LAMP sensitivity test of aphelenchoides simplicis; m: 100 bp Plus DNA ladder (Fermentas, USA); NC: double distilled water is used as a template; 100、10-1、10-2、10-3、10-4、10-5: the simple heterodera sinensis genomic DNA is used as a template.
FIG. 4 shows the result of LAMP-LFD sensitivity test of Heterodera simplex; NC: double distilled water is used as a template; 100、10-1、10-2、10-3、10-4、10-5: the simple heterodera sinensis genomic DNA is used as a template.
FIG. 5 shows the result of the LAMP-LFD sensitivity test of heterodera pekinensis;NC: double distilled water is used as a template; 100、10-1、10-2、10-3、10-4、10-5: the heterodera pekinensis genome DNA is used as a template.
FIG. 6 shows the result of the LAMP time optimization experiment for Heterodera simplex/Heterodera pekinensis; m: 100 bp Plus DNAsadeder (Fermentas, USA); NC: double distilled water is used as a template; 25. 30, 35, 40, 45, 50, 55 and 60 min: the sensitive genome DNA of the heterodera simplicissima/heterodera pekinensis is taken as a template.
FIG. 7 shows the result of the LAMP-LFD time optimization experiment of Heterodera simplex/Heterodera pekinensis; NC: double distilled water is used as a template; 25. 30, 35, 40, 45, 50, 55 and 60 min: the sensitive genome DNA of the heterodera simplicissima/heterodera pekinensis is taken as a template.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1
Establishment of method for detecting heterodera simplicissima/heterodera pekinensis by LAMP-LFD technology
1. Designing a primer: the primer is designed according to the published coding sequence of the rDNA-ITS2 (GenBank accession number: KU 991876) of the aphelenchoides simplicis/aphelenchoides pekinensis in NCBI, wherein the primer sequence is specifically as follows:
AniITS-F3：5’- GAATTGCAGACACATTGAGC -3’
AniITS-B3：5’- ATAGTAGATTCGGTGTTGACG -3’，
AniITS-FIP：5’- GTAATTCGACCCTCAGCCAGACACTAAGAATTCGAACGCACA -3’，
AniITS-BIP：5’- CTGTGAAGCATTCGGCAAGCAACCGCTCGTCATATTGTC -3’，
AniITS-LF：5’- TGCCATCGGGAATGAACC -3’，
AniITS-LB：5’- TTGCTGTTGTGTTGTTGGTG -3’，
probe AniITS-HP: 5'-GGTGAACTGTCTTCACGGTT-3' the flow of the air in the air conditioner,
wherein, the 5' end of AniITS-FIP is a biotin label; the 5' end of the probe AniITS-HP is labeled by fluorescein isothiocyanate.
2. Extracting sample DNA: treating single worms of each worm isolate with 70% ethanol, removing the ethanol, and then performing the steps according to the method of the tissue genome DNA extraction kit. Dissolved in 20. mu.L sterile ddH2O was used for LAMP validation.
3. LAMP reaction of Heterodera simplex/Heterodera pekinensis
And (2) performing LAMP amplification by using the specific primers designed in the step (1) and the genome DNA of the heterodera simplicissima/heterodera pekinensis as a template.
3.1 LAMP reaction system, the final concentration of each component is respectively: 0.2 mu mol/L of each of outer primers aniITS-F3 and aniITS-B3, 1.6 mu mol/L of each of inner primers aniITS-FIP and aniITS-BIP, 0.4 mu mol/L of each of loop primers aniITS-LF and aniITS-LB, 1.4 mmol/L of dNTPs, 20 mmol/L of Tris-HCl (pH 8.8), 10mmol/L of KCl, MgSO46.5mmol/L，(NH4)2SO410mmol/L, Triton X-1000.1%, 8U Bst DNA polymerase large fragment (NewEngland Biolabs) and 2 muL sample template, and adding double-distilled water to make the total volume of the reaction system be 25 muL;
3.2 LAMP reaction conditions: and carrying out amplification reaction on the reaction system, wherein the amplification reaction temperature is 63 ℃, and the amplification reaction time is 60 min.
4. Probe hybridization and LFD detection: and after amplification, adding 20 pmol of an AniITS-HP probe into the reaction system, incubating for 5 min at 63 ℃, hybridizing, adding 5 muL of hybridization solution into 100 muL of buffer, uniformly mixing, then immersing the LFD test strip into the buffer added with the hybridization solution for color development, and judging the amplification condition of the LAMP.
Example 2
Specificity determination for LAMP-LFD detection of Heterodera simplex/Heterodera pekinensis by using primers and probes of the invention
By using the designed specific primers and probes, performing LAMP-LFD reaction according to the steps 3 and 4 of the example 1 by using genome DNAs of heterodera simplex, heterodera pekoides, heterodera fascicularis, heterodera malabarica, typical heterodera, caenorhabditis clina, caenorhabditis grisea, heterodera inflata, heterodera, heterodera grisea, heterodera croplanus, heterodera fischeri, heterodera sinensis, heterodera fragrans, heterodera fischeri, heterodera sinensis, heterodera fischeri, and the like as templates, and verifying the specificity of the primers and the probes. As shown in FIGS. 1 and 2, the electrophoresis (FIG. 1) and LFD (FIG. 2) can only amplify the target band from the genomic DNA sample of the heterodera simplicissima/heterodera pekinensis, and the other samples have no amplified band, which indicates that the LAMP-LFD detection using the primers and the probe provided by the invention has good specificity.
Example 3
Sensitivity determination of LAMP-LFD detection of Aphelenchoides simply by using primers and probes of the invention
The genomic DNA of the nematode Ctenocephalides simply L3 was extracted by the method of step 2 of example 1, 10-fold gradient dilution was performed, and 10-fold selection was performed0、10-1、10-2、10-3、10-4、10-5: the LAMP-LFD reaction was performed according to the above steps 3 and 4 of example 1 using the simple Aphelenchoides genome as a template, and the sensitivity of the primers and the probe was verified, and double distilled water was used as a negative control. As a result, as shown in FIGS. 3 and 4, the LAMP-LFD detection using the primers and probes of the present invention had a sensitivity of 10 to a single worm genomic DNA extract-4And (4) doubling. The total volume of the simple heterodera cuneata L3 genome DNA extracting solution is 20 mu L, and 2 mu L is taken as a template to carry out LAMP reaction, so that the calculated sensitivity of LAMP-LFD is 10 of the genome DNA of a single worm-5The amplification product of LAMP was detected by agarose gel electrophoresis with the same sensitivity (FIG. 3).
Example 4
Sensitivity determination of heterodera pekinensis LAMP-LFD detection by using primers and probes of the invention
The genomic DNA of the heterodera pekinensis L3 was extracted by the method of step 2 of example 1, 10-fold gradient dilution was performed, and 10 was selected0、10-1、10-2、10-3、10-4、10-5: using the Heterodera splendens genome as a template, the LAMP-LFD reaction was performed in steps 3 and 4 of example 1And verifying the sensitivity of the primer and the probe, and using double distilled water as a negative control. As a result, as shown in FIG. 5, the LAMP-LFD detection using the primers and probes of the present invention had a sensitivity of 10 to a single worm genomic DNA extract-4And (4) doubling. Since the total volume of the heterodera peyronieri L3 genome DNA is 20 muL, 2 muL is taken as a template for LAMP reaction, and the calculated LAMP-LFD sensitivity is 10 of the genome DNA of a single worm-5And (4) doubling.
Example 5
Time optimization for LAMP-LFD detection of heterodera simplex/heterodera pekinensis by using primers and probes of the invention
Genomic DNA of heterodera simplex/heterodera pekinensis L3 was extracted by the method of step 2 of example 1, and LAMP-LFD reaction was performed according to step 3 and step 4 of example 1 by selecting genomic DNA of sensitive concentration as template, setting LAMP reaction time to 25, 30, 35, 40, 45, 50 and 60 min, respectively, and double distilled water as negative control. As a result, as shown in FIGS. 6 and 7, the optimal time for the LAMP reaction using the primers provided by the present invention was 40 min; after the LAMP product is added with the probe and incubated, the LAMP-LFD detection can be completed within 50 min.
Example 6
The LAMP-LFD technology of the invention is used for detecting the simple heterodera cruzi/heterodera pekinensis in the fish clinical samples
1. Extraction of genomic DNA from sample to be detected
Several strips of marine fish were purchased in the wholesale market of Ningbo, and the worms in the internal organs were dissected and isolated. The worms are morphologically identified after being washed with physiological saline water. Then, the genome DNA of the individual worms is extracted.
2. The LAMP reaction system preparation and reaction conditions were carried out in accordance with step 3 of example 1.
3. LFD was developed and the detection result was judged according to step 4 of example 1.
4. Identification by DNA sequencing method using PCR product
The results show that the worms in the clinical specimens tested are mostly of the species Heterodera (Table 1) according to morphological characteristics. The LAMP-LFD method is utilized to find that the infection rate of the aphelenchoides simplicis/the aphelenchoides pekinensis in clinical samples is 94%. The use of PCR in combination with sequencing led to consistent results (Table 1). The LAMP-LFD method established by the test can be used for the field detection of the simple heterodera cuneata/heterodera pekinensis.
TABLE 1 detection results of heterodera simplicissima/heterodera pekinensis in fish samples by microscopic observation, LAMP-LFD and PCR combined DNA sequencing method
Note: "+" indicates a positive test result; "-" indicates negative result.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that such changes, modifications, additions and substitutions are within the true spirit of the invention.
Sequence listing
<110> Ningbo university
<120> primer and probe sequences for LAMP-LFD detection of Heterodera simplex/Heterodera pekinensis
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Claims (1)
1. The application of a group of primers and probes in preparing the LAMP-LFD detection reagent for detecting the heterodera simplicissima and the heterodera pekinensis is characterized in that three pairs of primer sequences and one probe sequence of the LAMP-LFD are designed according to the coding sequence of an internal transcribed spacer rDNA-ITS2 sequence of the ribose of the heterodera simplicissima/the heterodera pekinensis, and the primer sequences and the probe sequences are specifically as follows:
AniITS-F3：5’-GAATTGCAGACACATTGAGC-3’，
AniITS-B3：5’-ATAGTAGATTCGGTGTTGACG-3’，
AniITS-FIP：5’-GTAATTCGACCCTCAGCCAGACACTAAGAATTCGAACGCACA-3’，
AniITS-BIP：5’-CTGTGAAGCATTCGGCAAGCAACCGCTCGTCATATTGTC-3’，
AniITS-LF：5’-TGCCATCGGGAATGAACC-3’，
AniITS-LB：5’-TTGCTGTTGTGTTGTTGGTG-3’，
probe AniITS-HP: 5'-GGTGAACTGTCTTCACGGTT-3' the flow of the air in the air conditioner,
wherein, the 5' end of AniITS-FIP is a biotin label; the 5' end of the probe AniITS-HP is labeled by fluorescein isothiocyanate.
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| CN107245521A (en) | 2017-10-13 |
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