Multiplex PCR primer combination, kit and detection method for detecting echinococcus species

Abstract

This invention discloses a multiplex PCR primer combination, kit, and detection method for detecting Echinococcus granulosus. The multiplex PCR primer combination includes a second primer combination and a third primer combination: the second primer combination consists of upstream and downstream primers specific to Echinococcus multilocularis, with sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively; the third primer combination consists of upstream and downstream primers specific to Echinococcus shiquensis, with sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. This invention saves more time than conventional PCR, is more cost-effective than quantitative real-time PCR, and has lower requirements for operators and equipment, showing great promise for the detection and control of echinococcosis.

Description

Technical Field

[0001] This invention belongs to the field of parasite detection technology, specifically a multiplex PCR primer combination, kit, and detection method for detecting Echinococcus tapeworm species. Background Technology

[0002] Echinococcosis, commonly known as echinococcosis, is caused by the larvae Echinococcus. Echinococcus spp. Echinococcus larvae (hydatid larvae) parasitize the liver, lungs, brain, and other organs of mammals such as humans, sheep, and cattle, causing zoonotic parasitic diseases that seriously endanger livestock production and human health. In my country, it is classified as a Class C infectious disease and a Class II animal disease, and is a key parasitic disease for prevention and control.

[0003] Of the known Echinococcus tapeworms, Echinococcus granulosus ( Echinococcus granulosus s.s. ), Echinococcus multilocularis ( Echinococcus multilocularis ), Echinococcus shiquensis ( Echinococcus shiquicus Echinococcus granulosus is a major prevalent echinococcosis species in my country's agricultural and pastoral areas. Among them, *Echinococcus granulosus* larvae cause cystic echinococcosis, *Echinococcus multilocularis* larvae cause alveolar echinococcosis, and *Echinococcus shiquensis*, a species endemic to my country, is mainly prevalent in the western Sichuan Plateau and other regions. The overlapping prevalence areas of these three species pose a significant challenge to accurate detection and control. Currently, there are 370 counties in my country where echinococcosis is prevalent, distributed across 9 provinces (autonomous regions). The annual losses to livestock and the burden of medical care caused by this disease are extremely heavy.

[0004] The detection methods for Echinococcus granulosus infection mainly fall into four categories: etiological detection, imaging detection, immunological detection, and molecular biological detection. Etiological detection requires microscopic observation of the worm, eggs, or protoscolex, which is cumbersome, time-consuming, and demands high technical skills from the personnel. Furthermore, it has extremely low detection rates for early and mild infections, making it difficult to meet practical testing needs. Imaging detection (such as ultrasound, CT, and MRI) is primarily used for the clinical diagnosis of echinococcosis, but it struggles to diagnose early infections and atypical imaging features. It also has poor applicability to laboratory screening and on-site testing, and is highly dependent on equipment. Immunological detection, with enzyme-linked immunosorbent assay (ELISA) as a common method, is relatively simple to perform, but its specificity is insufficient due to factors such as antigen quality and individual differences in immune response. It also cannot specifically distinguish between the three types of Echinococcus granulosus, and is prone to cross-reactions, making it difficult to meet the requirements for accurate detection. In molecular biology detection, conventional PCR has the advantages of high specificity and high sensitivity. However, conventional PCR can only detect one type of Echinococcus tapeworm at a time. If three target worms need to be detected, three PCR reactions need to be performed separately. The operation steps are cumbersome, the detection cycle is long, and the consumption of reagents and samples is large, resulting in low efficiency. It cannot meet the needs of large-scale screening and rapid on-site detection. Summary of the Invention

[0005] In view of the shortcomings of existing detection technologies, such as low detection efficiency and inability to distinguish between two or three main Echinococcus tapeworms at the same time, it is necessary to develop a method that can simultaneously and accurately detect Echinococcus multilocularis, Echinococcus shiquensis, and even Echinococcus granulosus.

[0006] In a first aspect, the present invention provides a multiplex PCR primer set for detecting Echinococcus tapeworm species, comprising a second primer set and a third primer set: The second primer combination consists of a *Echinococcus multilocularis*-specific upstream primer (F2) and downstream primer (R2), the sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The third primer combination consists of a specific upstream primer (F3) and a downstream primer (R3) for Echinococcus shiquense, with sequences shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0007] In some embodiments, the multiplex PCR primer combination further includes a first primer combination; the first primer combination is a *Echinococcus granulosus*-specific upstream primer (F1) and downstream primer (R1), the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0008] Secondly, the present invention provides a kit for detecting Echinococcus tapeworm species, comprising the multiplex PCR primer combination described in the first aspect.

[0009] In some implementations, the kit also includes 2×Premix Taq enzyme premix.

[0010] Thirdly, the present invention provides a method for detecting Echinococcus tapeworm species, which uses the multiplex PCR primer combination described in the first aspect for multiplex PCR.

[0011] In some implementation schemes, the PCR reaction conditions are: 95°C pre-denaturation for 5 min; 95°C denaturation for 15 sec, 56°C annealing for 30 sec, 72°C extension for 30 sec, 35 cycles; 72°C final extension for 10 min.

[0012] In some implementation schemes, the PCR reaction system consists of: 1 μL DNA template, 12.5 μL 2×Premix Taq enzyme premix, 0.8 μL each of 10 μM Echinococcus multilocularis-specific upstream and downstream primers, 1 μL each of 10 μM Echinococcus shiquensis-specific upstream and downstream primers, and the remainder is brought to a total reaction volume of 25 μL with ddH2O or dH2O.

[0013] In some implementation schemes, the PCR reaction system comprises: 1 μL DNA template, 12.5 μL 2×Premix Taq enzyme premix, 1-1.2 μL each of 10 μM *Echinococcus granulosus*-specific upstream and downstream primers; 0.8 μL each of 10 μM *Echinococcus multilocularis*-specific upstream and downstream primers, 1 μL each of 10 μM *Echinococcus shiquensis*-specific upstream and downstream primers, with the remainder made up to a total reaction volume of 25 μL using ddH2O or dH2O.

[0014] Furthermore, the PCR reaction system contains 1 μL each of the 10 μM *Echinococcus granulosus*-specific upstream and downstream primers.

[0015] In some implementations, the PCR products are detected by gel electrophoresis: if a band appears at approximately 230 bp, it is *Echinococcus granulosus*; if a band appears at approximately 530 bp, it is *Echinococcus multilocularis*; and if a band appears at approximately 420 bp, it is *Echinococcus shiquensis*.

[0016] In some implementations, the PCR products are detected by gel electrophoresis; the gel electrophoresis is 2% agarose gel electrophoresis, and the electrophoresis conditions are 120V for 40 minutes.

[0017] Compared with the prior art, the main advantages of the present invention are as follows: 1. Some implementation schemes enable simultaneous detection of three Echinococcus tapeworm species, addressing the shortcomings of existing technologies: overcoming the limitations of conventional PCR, which can only detect one type of Echinococcus tapeworm at a time and is cumbersome to operate. Simultaneously covering Echinococcus granulosus, Echinococcus multilocularis, and Echinococcus shiquensis, these schemes are adapted to the epidemiological characteristics of Echinococcus tapeworms in my country, meeting the need for accurate differentiation of the three prevalent species.

[0018] 2. Improve detection efficiency and reduce detection costs: By adopting multiplex PCR technology, some implementation schemes can amplify three target insect species in the same reaction system without having to perform PCR reactions three times. This reduces operation steps, shortens the detection cycle, and reduces reagent and sample consumption, making it suitable for large-scale screening and on-site detection scenarios.

[0019] This invention saves more time than conventional PCR and is more cost-effective than quantitative real-time PCR. It also has low requirements for operators and equipment and has great application prospects in the field of echinococcosis detection and control.

[0020] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a diagram showing the detection results of Example 2 of the present invention. The lanes are as follows: 1. *Echinococcus granulosus*; 2. *Echinococcus multilocularis*; 3. *Echinococcus scutellatus*; 4. *Echinococcus granulosus* + *Echinococcus multilocularis*; 5. *Echinococcus granulosus* + *Echinococcus scutellatus*; 6. *Echinococcus multilocularis* + *Echinococcus scutellatus*; M. DNA marker.

[0023] Figure 2 This is a diagram showing the specificity test results of Experiment Example 1 of the present invention. The lanes are as follows: 1. *M. DNA Msrker*; 2. *Echinococcus granulosus*; 3. *Echinococcus multilocularis*; 4. *Taenia obeseis*; 5. *Taenia chainensis*; 6. *Taenia asiatica*; 7. *Taenia vesicularis*; 8. *Taenia gigantea*; 9. Blank control. Detailed Implementation

[0024] To facilitate understanding by those skilled in the art, some terms appearing in this document are explained and clarified.

[0025] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural references.

[0026] In this document, unless otherwise stated, the terms “comprising,” “including,” or “containing” mean that the listed technical features are included, but do not exclude the inclusion of other technical features.

[0027] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0028] Example 1 Acquisition and synthesis of primer combinations To establish a multiplex PCR method for detecting three Echinococcus granulosus species—Echinococcus granulosus, Echinococcus multilocularis, and Echinococcus shiquensis—this experiment selected the mitochondrial genes of Echinococcus granulosus (GenBank accession number: AF297617.1), Echinococcus multilocularis (GenBank accession number: NC_000928.2), and Echinococcus shiquensis (GenBank accession number: NC_009460.1) as reference sequences and designed corresponding oligonucleotide primer sequences. The primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Table 1 lists a preferred set of primer sequences.

[0029] Table 1. Primer sequences Example 2 Establishment of multiplex PCR reaction system and method (1) Genomic DNA was extracted from the sample to be tested to obtain a DNA template, which was stored at -20℃ for later use and removed before testing.

[0030] (2) After centrifuging the dry powder of each primer synthesized in Example 1, dissolve it in dH2O to 10 μM. Mix the DNA template, 2×Premix Taq enzyme premix, primers and dH2O obtained in step (1) according to Table 2. All operations should be performed in an environment free from nucleic acid contamination.

[0031] Table 2. Reaction System (3) Place the PCR reaction system configured in step (2) into a PCR instrument and set the reaction conditions as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 sec, 56℃ annealing for 30 sec, 72℃ extension for 30 sec, 35 cycles; 72℃ final extension for 10 min.

[0032] (4) After the reaction in step (3) is completed, the PCR amplification products are electrophoresed in a 2% agarose gel at 120V for 40 minutes. After electrophoresis, the results are observed under UV light. If a band appears at approximately 230bp, it is *Echinococcus granulosus*; if a band appears at approximately 530bp, it is *Echinococcus multilocularis*; if a band appears at approximately 420bp, it is *Echinococcus shiquensis*. Figure 1 .

[0033] Experimental Example 1 Specificity test of multiplex PCR method The multiplex PCR method established above was used to detect Echinococcus granulosus, Echinococcus multilocularis, Echinococcus shiquensis, and Echinococcus obliquus. Taenia saginata ), chain tapeworm (Taenia solium Asian tapeworm ( Taenia asiatica ), Vesicular tapeworm ( Taenia hydatigena Giant neck tapeworm ( Taenia taeniaeformis DNA samples were tested to verify the specificity of the multiplex PCR method.

[0034] The results are as follows Figure 2 As shown, the multiplex PCR method established in this invention can specifically detect *Echinococcus granulosus*, *Echinococcus multilocularis*, and *Echinococcus shiquensis*, with specific amplification bands appearing at approximately 230bp, 530bp, and 420bp, respectively. No amplification bands were observed for other pathogens such as *Taenia obliquus*, *Taenia chainratus*, *Taenia asiatica*, *Taenia vesicularia*, and *Taenia gigantea*. This indicates that the multiplex PCR method has good specificity.

[0035] The above experiments demonstrate that this invention, as a rapid, economical, and low-requirement multiplex PCR detection method for operators and equipment, can accurately detect three major prevalent Echinococcus species in my country: Echinococcus granulosus, Echinococcus multilocularis, and Echinococcus shiquensis. It has excellent application prospects in the field of echinococcosis detection and control.

[0036] The above embodiments and accompanying drawings are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A multiplex PCR primer set for detecting Echinococcus tapeworm species, characterized in that, Including the second primer combination and the third primer combination: The second primer combination consists of Echinococcus multilocularis-specific upstream and downstream primers, the sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The first primer combination consists of upstream and downstream primers specific to Echinococcus shiquensis, and their sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

2. The multiplex PCR primer combination for detecting Echinococcus tapeworm species according to claim 1, characterized in that, It also includes a first primer combination; the first primer combination is a *Echinococcus granulosus*-specific upstream and downstream primers, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

3. A kit for detecting Echinococcus tapeworm species, characterized in that, Includes the multiplex PCR primer combination as described in claim 1 or 2.

4. The kit for detecting Echinococcus tapeworm species according to claim 3, characterized in that, It also includes 2×PremixTaq enzyme premix.

5. A method for detecting Echinococcus tapeworm species, characterized in that, Multiplex PCR was performed using the multiplex PCR primer combination described in claim 1 or 2.

6. The method for detecting Echinococcus tapeworm species according to claim 5, characterized in that, The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 sec, 56℃ annealing for 30 sec, 72℃ extension for 30 sec, for 35 cycles; and 72℃ final extension for 10 min.

7. The method for detecting Echinococcus tapeworm species according to claim 5, characterized in that, Multiplex PCR was performed using the primer combination described in claim 1. The PCR reaction system consisted of: 1 μL DNA template, 12.5 μL 2×Premix Taq enzyme premix, 0.8 μL each of the 10 μM *Echinococcus multilocularis*-specific upstream and downstream primers, 1 μL each of the 10 μM *Echinococcus shiquensis*-specific upstream and downstream primers, and the remainder was brought to a total reaction volume of 25 μL with ddH2O or dH2O.

8. The method for detecting Echinococcus tapeworm species according to claim 5, characterized in that, Multiplex PCR was performed using the primer combination described in claim 2. The PCR reaction system consisted of: 1 μL DNA template, 12.5 μL 2×Premix Taq enzyme premix, 1-1.2 μL each of the 10 μM *Echinococcus granulosus*-specific upstream and downstream primers, 0.8 μL each of the 10 μM *Echinococcus multilocularis*-specific upstream and downstream primers, 1 μL each of the 10 μM *Echinococcus shiquensis*-specific upstream and downstream primers, and the remainder was brought to a total reaction volume of 25 μL with ddH2O or dH2O.

9. The method for detecting Echinococcus tapeworm species according to claim 8, characterized in that, PCR products were detected by gel electrophoresis: if a band appeared at around 230 bp, it was *Echinococcus granulosus*; if a band appeared at around 530 bp, it was *Echinococcus multilocularis*; and if a band appeared at around 420 bp, it was *Echinococcus shiquensis*.

10. The method for detecting *Echinococcus granulosus* species according to any one of claims 5 to 9, characterized in that, The PCR products were detected by gel electrophoresis; the gel electrophoresis was 2% agarose gel electrophoresis, and the electrophoresis conditions were 120V for 40min.

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