ARMS-PCR primer group, kit and method for fluke flakes

Abstract

The invention relates to an ARMS-PCR primer group, a kit and a method for fluke flakes, and relates to the field of molecular biology and parasitology. The ARMS-PCR primer group can overcome the defects of an existing parasite detection and typing identification method, whether a person / animal to be detected is infected with parasite or not can be judged simply, rapidly and accurately by utilizing the primer group, and the type of the infected parasite can be identified, so that a good basis is provided for subsequent treatment or medication.

Description

Technical Field

[0001] The invention relates to the fields of molecular biology and parasitology, and in particular to an ARMS-PCR primer set, a kit and a method for fasciola. Background Art

[0002] Fascioliasis is a foodborne zoonosis caused by trematodes of the genus Fasciola (1758) of the family Fasciolidae that parasitize the hepatobiliary ducts of ruminants such as cattle and sheep and humans, leading to pathological damage to the liver and gallbladder of the host. Fascioliasis causes significant economic losses to the animal husbandry industry and is also an important public health issue. At present, at least 2.4 million people are infected with fascioliasis worldwide, and 91 million people are at risk of infection. There are four species in the genus Fascioliasis: F. hepatica, F. gigantica, F. jacksoni, and F. nyanzae. F. hepatica and F. gigantica are widely prevalent in my country and are the most harmful to animals and humans. Among them, F. hepatica is mainly prevalent in the northern region of my country and is the dominant species in the temperate zone, while F. gigantica is mainly prevalent in the southern region of my country and is the dominant species in the tropical and subtropical regions. Since Fasciola hepatica and Fasciola gigantea are very similar in morphology, and in recent years, hybrid Fasciola species have even appeared in many areas of Asia and Africa, which poses a challenge to the traditional morphological classification and identification of Fasciola species, and also increases the difficulty of identifying Fasciola species. Since different species of Fasciola differ in terms of antigenicity, immunogenicity, pathogenicity and drug sensitivity, the correct identification of Fasciola species and the accurate understanding of their phenotypic and molecular characteristics are helpful to accurately identify the cause of the disease, achieve effective prevention and control, and provide a reference for vaccine development.

[0003] For a long time, the detection and classification of Fasciola have been mainly carried out according to the characteristics such as adult morphology, life history and epidemiology, but due to the large individual differences in the species of Fasciola, the same worm species is different because of the difference in host species and host response, causing the different individual morphologies of the same worm species to be irregular. In recent years, the development of molecular biology techniques has provided new approaches for the identification of Fasciola. For example, RFLP (restriction fragment length polymorphism) analysis method can be applied to the detection and identification of point mutations. However, the usual steps of this method are complicated, and operation is cumbersome, and specific conditions are required, and the limitation is strong, which limits its widespread application in practice. Summary of the invention

[0004] In view of the above problems, the present invention provides an ARMS-PCR primer set for Fasciola, which can solve the shortcomings of existing methods for detecting and typing Fasciola. The primer set can not only be used to judge whether a person to be tested is infected with Fasciola in a simple, rapid and highly accurate manner, but also can identify the type of infected Fasciola, thereby providing a good basis for subsequent treatment or medication.

[0005] In order to achieve the above object, the present invention provides an ARMS-PCR primer set for Fasciola, comprising an outer primer and an inner primer;

[0006] The outer primers include:

[0007] Outer-F:5'-TTGCGCTGATTACGTCCCTG-3'(SEQ NO.9);

[0008] Outer-R:5'-TTGGCTGCGCTCTTCATCGAC-3'(SEQ NO.10);

[0009] The inner primers include:

[0010] Inner-F:5'-CGCCCTATGAACTGTTTCATTACTAGAT-3'(SEQ NO.11);

[0011] Inner-R: 5'-GCCATTCAGTACCACTTTAACAGTGTTAT-3' (SEQ NO. 12).

[0012] Traditional detection of Fasciola infection mainly relies on blood tests and etiological examinations, while the morphological identification method of Fasciola mainly relies on naked eye observation. However, traditional infection detection methods are prone to missed diagnosis when the number of parasites is small, and misdiagnosis is easy to occur due to the similarity between the eggs of Fasciola hepatica and those of Fasciola gingivalis, Fasciola gigantea and Fasciola trichomoniasis. Although the diagnosis can be confirmed by direct surgical laparotomy or bile duct surgery, this method will cause great trauma to the personnel / animals being tested and has obvious limitations. Traditional morphological identification methods are difficult to accurately distinguish these tiny morphological differences by naked eye observation because different types of Fasciola are very similar in morphology, especially in the larval stage. Morphological identification requires the operator to have rich experience and superb skills to make correct judgments on morphological characteristics, which creates a high degree of dependence and subjectivity on the accuracy of the identification results. When faced with samples with vague or atypical morphological characteristics, misjudgment may also occur, resulting in unreliable identification results, a high risk of misjudgment, and difficulty in accurately distinguishing morphologically similar Fasciola species and their heterozygotes.

[0013] The limitation of the RFLP method is that it relies on the presence of specific restriction endonuclease sites near the mutation site. This means that the RFLP technique can only be effectively applied when there are suitable restriction endonuclease sites around the mutation site. If there is a lack of suitable restriction endonuclease sites in the target sequence, the RFLP method will not be able to achieve effective gene mutation detection. Therefore, the scope of application of the RFLP method is greatly limited and cannot meet all gene detection needs.

[0014] ARMS-PCR is a highly specific PCR technology specifically used to detect single nucleotide polymorphisms (SNPs). Since TaqDNA polymerase lacks 3'-5' exonuclease activity, mismatches at the 3' end of the PCR primer will lead to a reduction in amplification products. Therefore, suitable primers can be designed for known SNP sites to detect infection conditions and genotyping.

[0015] The invention also provides an ARMS-PCR reaction system for Fasciola, comprising the ARMS-PCR primer set.

[0016] In one embodiment, the working concentration of the inner primer is 10-14% by volume, and the working concentration of the outer primer is 4-8% by volume.

[0017] The working concentration is the concentration at which the inner primers and outer primers can achieve the expected effect when used. It can be understood that when preparing the ARMS-PCR reaction system, those skilled in the art can prepare a mother solution or a stock solution with a higher concentration, and then dilute it when used. The mother solution or the stock solution and its concentration are all within the protection scope of the present invention.

[0018] In one embodiment, the reaction conditions of the ARMS-PCR reaction system include: pre-denaturation at 95°C for 3 min; 20 cycles of denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 10 min.

[0019] The present invention also provides a kit for Fasciola, comprising the ARMS-PCR primer set or the ARMS-PCR reaction system.

[0020] The present invention also provides a method for identifying Fasciola, comprising the following steps: extracting DNA of a sample to be tested, using the kit for detection to obtain an amplification product, performing electrophoresis on the amplification product, and identifying the type of Fasciola in the sample to be tested according to the electrophoresis result.

[0021] In one embodiment, the type identification includes: when the electrophoresis result includes a 680 bp band and a 509 bp band, the Fasciola is Fasciola gigantea;

[0022] When the electrophoresis result includes a 680 bp band and a 227 bp band, the fasciola is Fasciola hepatica;

[0023] When the electrophoresis result includes 680 bp, 509 bp and 227 bp bands, the Fasciola is a heterozygote of Fasciola hepatica and Fasciola gigantea.

[0024] The present invention also provides a method for detecting Fasciola for non-diagnostic purposes, comprising the following steps: extracting DNA of a sample to be tested, using the kit for detection to obtain an amplification product, performing electrophoresis on the amplification product, and judging whether the sample to be tested contains Fasciola according to the electrophoresis result.

[0025] In one embodiment, the judgment includes: if the electrophoresis result includes at least one of a 680 bp band, a 509 bp band or a 227 bp band, then the sample to be tested is infected with Fasciola.

[0026] The present invention also provides the use of the ARMS-PCR primer set in preparing a product for identifying the type of Fasciola and / or detecting Fasciola.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The ARMS-PCR primer set, kit and method for Fasciola of the present invention can solve the shortcomings of existing methods for detecting and typing Fasciola. The primer set can not only be used to simply, quickly and accurately determine whether a person or animal to be tested is infected with Fasciola, but also can identify the type of infected Fasciola, thereby providing a good basis for subsequent treatment or medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Amplification results of ARMS-PCR primer combinations designed for different SNP sites in Example 1;

[0030] Figure 2 Schematic diagram of the best primer combination obtained in Example 1;

[0031] Figure 3 This is a diagram of the amplification results of some ARMS-PCR amplification reaction systems in the optimization experiment in Example 1;

[0032] Figure 4 The electrophoresis diagram of the best primer combination and the best ARMS-PCR amplification reaction system in Example 1 is shown. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0036] Example 1

[0037] The inventors found in the experimental process that the difficulty of using ARMS-PCR to detect Fasciola mainly lies in the complexity of primer design, the selection of SNP sites and the design of mismatched bases, as well as the optimization of PCR reaction system and program.

[0038] 1. First of all, primer design is the key to ARMS-PCR technology, which requires extremely high specificity and accuracy. The 3' end of each primer must completely match the target SNP site to ensure that effective extension can only be performed when the primer terminal base completely matches the template DNA. In order to increase specificity, it is also necessary to introduce mismatched bases near the 3' end of the primer, which further increases the difficulty of design. To ensure that the primers can effectively distinguish between Fasciola gigantea, Fasciola hepatica and their heterozygotes, a lot of bioinformatics analysis and experimental verification are required.

[0039] Therefore, the present invention firstly accurately analyzes the gene sequence of Fasciola through bioinformatics, selects multiple different SNP sites, and designs multiple sets of ARMS-PCR primer combinations for different SNP sites for preliminary screening, wherein SNP sites with high representativeness and specificity are preferentially selected to ensure that different types of Fasciola and their heterozygotes can be accurately distinguished. The following shows some SNP sites and the corresponding ARMS-PCR primer combinations, and the amplification results are shown as follows: Figure 1 .

[0040] SNP site 1:

[0041] Outer-F:5'-TTGCGCTGATTACGTCCCTG-3'(SEQ NO.1);

[0042] Outer-R:5'-TTGGCTGCGCTCTTCATCGAC-3'(SEQ NO.2);

[0043] Inner-F:5'-CGGAAGGATCATTACCTGAAAATCTACTATC-3'(SEQ NO.3);

[0044] Inner-R:5'-TCACACGTGTATCGCTTGTCTA-3'(SEQ NO.4)。

[0045] SNP 2:

[0046] Outer-F:5'-TTGCGCTGATTACGTCCCTG-3'(SEQ NO.5);

[0047] Outer-R:5'-TTGGCTGCGCTCTTCATCGAC-3'(SEQ NO.6);

[0048] Inner-F:5'-GGAGAACGGGTTGTACTGCTAC-3'(SEQ NO.7);

[0049] Inner-R:5'-CTCTTTAAGCCTAGCACTACCAAGCA-3'(SEQ NO.8)。

[0050] SNP 3:

[0051] Outer-F:5'-TTGCGCTGATTACGTCCCTG-3'(SEQ NO.9);

[0052] Outer-R:5'-TTGGCTGCGCTCTTCATCGAC-3'(SEQ NO.10);

[0053] Inner-F:5'-CGCCCTATGAACTGTTTCATTACTAGAT-3'(SEQ NO.11);

[0054] Inner-R:5'-GCCATTCAGTACCACTTTAACAGTGTTAT-3'(SEQ NO.12)。

[0055] SNP 4:

[0056] Outer-F:5'-TTGCGCTGATTACGTCCCTG-3'(SEQ NO.13);

[0057] Outer-R:5'-TTGGCTGCGCTCTTCATCGAC-3'(SEQ NO.14);

[0058] Inner-F: 5'-CTACATTTACACTGTTAAAGTGGGAC-3' (SEQ NO. 15);

[0059] Inner-R: 5'-GGCAAAGAATGGCAAGCCATTTAA-3' (SEQ NO. 16).

[0060] Design highly specific primers and introduce mismatched bases to improve specificity and avoid nonspecific amplification ( Figure 2 ). And through multiple optimization and experimental verification, the best primer combination was screened out.

[0061] Second, the optimization of ARMS-PCR reaction is another major difficulty. To amplify multiple target sites simultaneously in one reaction system, it is necessary to balance the amplification efficiency of each pair of primers and avoid mutual interference between primers. Reaction conditions (such as primer concentration, annealing temperature, and cycle parameters) need to be optimized through repeated experiments to ensure the best amplification effect for each target site. Mutual interference between different primers and differences in amplification efficiency may lead to unsatisfactory amplification effects for certain target sites, affecting the accuracy of the test results.

[0062] The selection of appropriate SNP sites and the introduction of mismatch bases are also crucial. The SNP sites must be representative and specific to accurately distinguish different types of Fasciola and their heterozygotes. The design of mismatch bases needs to be precise to ensure that only the target SNP site is amplified during the amplification reaction without nonspecific amplification. Improper selection and design can lead to false positive or false negative results.

[0063] Therefore, a systematic optimization experiment was conducted to determine the best ARMS-PCR reaction conditions. By continuously adjusting the primer concentration, annealing temperature and cycle parameters, the following shows some ARMS-PCR amplification reaction systems and their amplification results in the optimization experiment ( Figure 3 ) to ensure that the amplification efficiency of each target site in the reaction system is optimal and to avoid mutual interference between primers.

[0064] Table 1 ARMS-PCR amplification reaction system ( Figure 3 Top left)

[0065]

[0066] The reaction conditions of the reaction system were as follows: pre-denaturation at 95°C for 3 min; 20 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 10 min.

[0067] Table 2 ARMS-PCR amplification reaction system ( Figure 3 Top right)

[0068]

[0069] The reaction conditions of the reaction system were as follows: pre-denaturation at 95°C for 3 min; 20 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 10 min.

[0070] Table 3 ARMS-PCR amplification reaction system ( Figure 3 Lower left)

[0071]

[0072]

[0073] The reaction conditions of the reaction system were as follows: pre-denaturation at 95°C for 3 min; 20 cycles of denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 10 min.

[0074] Table 4 ARMS-PCR amplification reaction system ( Figure 3 Lower right)

[0075]

[0076] The reaction conditions of the reaction system were as follows: pre-denaturation at 95°C for 3 min; 20 cycles of denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 10 min.

[0077] 3. Through the above-mentioned large number of experiments, the inventors designed four primers based on the ARMS-PCR reaction: two inner primers (one specific to the allele of Fasciola gigantea and the other specific to the allele of Fasciola hepatica) and two outer primers (universal primers for Fasciola). The 3' end of the inner primer is completely complementary to the target SNP site, and one or more mismatched bases are deliberately introduced near the 3' end to enhance specificity. Through this design, when the 3' terminal base of the inner primer is completely matched with the template DNA, DNA polymerase can effectively extend and amplify, thereby achieving specific amplification, and the infection status and genotype of Fasciola can be efficiently and clearly judged by observing the amplified product by gel electrophoresis.

[0078] 1. Design ARMS-PCR primers.

[0079] ARMS-PCR primers were designed based on the gene sequence published in NCBI using Snapgene software, and the following primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Table 5):

[0080] Table 5 Specific primer sequences

[0081]

[0082] In order to increase the specificity of the primer, mismatched bases were added to the primer sequence. The 3' terminal base of Inner-F was completely complementary to the allele sequence of Fasciola hepatica, and a mismatched base C→G was added at the third to last position of the 3' terminal; the 3' terminal base of primer Inner-R was completely complementary to the allele sequence of Fasciola gigantea, and a mismatched base A→T was added at the third to last position of the 3' terminal.

[0083] 2. ARMS-PCR amplification

[0084] Establish an ARMS-PCR amplification reaction system (Table 6) and add the sample DNA to be tested:

[0085] Table 6 ARMS-PCR amplification reaction system

[0086]

[0087] ARMS-PCR amplification reaction conditions: pre-denaturation at 95°C for 3 min; followed by 20 cycles of denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 1 min; and finally final extension at 72°C for 10 min.

[0088] Agarose gel electrophoresis: Take 20 μl of the amplified product and run it on a 0.7% agarose gel at 120 V for 25 min, and observe the gel imaging results.

[0089] The imaging results are judged: the results are as follows Figure 4 As shown, a 680bp band and a 509bp band appeared in lane 1, indicating that the test sample was a Fasciola gigantea sample; a 680bp band and a 227bp band appeared in lane 2, indicating that the test sample was a Fasciola hepatica sample; a 680bp band, a 509bp band and a 227bp band appeared in lane 3, indicating that the test sample was a heterozygous sample of Fasciola hepatica and Fasciola gigantea; lane 4 was a negative control.

[0090] Example 2

[0091] 1. A method for identifying Fasciola.

[0092] The identification was performed by using the amplification primer set preferably obtained in Example 1 and the ARMS-PCR amplification reaction system (as shown in Table 5 and Table 6) to form a kit.

[0093] The method comprises: extracting DNA of a sample to be tested, using the above kit to detect, obtaining an amplified product, performing electrophoresis on the amplified product, and distinguishing the type of the Fasciola in the sample to be tested according to the electrophoresis result:

[0094] When the electrophoresis result includes a 680 bp band and a 509 bp band, the fasciola is a large fasciola;

[0095] When the electrophoresis result includes a 680 bp band and a 227 bp band, the fasciola is Fasciola hepatica;

[0096] When the electrophoresis result includes 680 bp, 509 bp and 227 bp bands, the Fasciola is a heterozygote of Fasciola hepatica and Fasciola gigantea.

[0097] 2. A method for detecting Fasciola.

[0098] The detection was performed by using the amplification primer set preferably obtained in Example 1 and the ARMS-PCR amplification reaction system (as shown in Table 5 and Table 6) to form a kit.

[0099] The detection method comprises the following steps: extracting DNA of a sample to be tested, using the kit to perform detection to obtain an amplified product, performing electrophoresis on the amplified product, and judging whether the sample to be tested contains Fasciola according to the electrophoresis result:

[0100] If the electrophoresis result includes at least one of a 680 bp band, a 509 bp band or a 227 bp band, the sample to be tested is infected with Fasciola.

[0101] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0102] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An ARMS-PCR primer set for Fasciola, characterized in that: Including outer primers and inner primers; The outer primers include: Outer-F:5'-TTGCGCTGATTACGTCCCTG-3'(SEQ NO.9); Outer-R:5'-TTGGCTGCGCTCTTCATCGAC-3'(SEQ NO.10); The inner primers include: Inner-F:5'-CGCCCTATGAACTGTTTCATTACTAGAT-3'(SEQ NO.11); Inner-R: 5'-GCCATTCAGTACCACTTTAACAGTGTTAT-3' (SEQ NO. 12).

2. An ARMS-PCR reaction system for Fasciola, characterized in that: Comprising the ARMS-PCR primer set described in claim 1.

3. The ARMS-PCR reaction system according to claim 2, characterized in that: The working concentration of the inner primer is 10-14% by volume, and the working concentration of the outer primer is 4-8% by volume.

4. The ARMS-PCR reaction system according to claim 2, characterized in that: The reaction conditions of the ARMS-PCR reaction system include: pre-denaturation at 95°C for 3 minutes; 20 cycles of denaturation at 95°C for 30 seconds, annealing at 53°C for 30 seconds, and extension at 72°C for 1 minute; and final extension at 72°C for 10 minutes.

5. A kit for Fasciola, characterized in that: The method comprises the ARMS-PCR primer set according to claim 1 or the ARMS-PCR reaction system according to any one of claims 2 to 4.

6. A method for identifying Fasciola, characterized in that: The following steps are involved: Extract DNA from the sample to be tested, use the kit described in claim 5 for detection, obtain an amplified product, perform electrophoresis on the amplified product, and identify the type of Fasciola in the sample to be tested based on the electrophoresis result.

7. The identification method according to claim 6, characterized in that: The type identification includes: when the electrophoresis result includes a 680 bp band and a 509 bp band, the fasciola is a large fasciola; When the electrophoresis result includes a 680 bp band and a 227 bp band, the fasciola is Fasciola hepatica; When the electrophoresis result includes 680 bp, 509 bp and 227 bp bands, the Fasciola is a heterozygote of Fasciola hepatica and Fasciola gigantea.

8. A method for detecting Fasciola for non-diagnostic purposes, characterized in that: The following steps are involved: Extract DNA from the sample to be tested, use the kit described in claim 5 to detect, obtain an amplified product, perform electrophoresis on the amplified product, and determine whether the sample to be tested contains Fasciola based on the electrophoresis result.

9. The detection method according to claim 8, characterized in that: The judgment includes: if the electrophoresis result includes at least one of a 680 bp band, a 509 bp band or a 227 bp band, then the sample to be tested is infected with Fasciola.

10. Use of the ARMS-PCR primer set according to claim 1 in preparing a product for identifying the type of Fasciola and / or detecting Fasciola.

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