阅读说明：本技术 用于鉴定多房棘球绦虫引物对、引物探针组以及他们的应用 (Primer pair and primer probe set for identifying echinococcus multilocularis and application of primer pair and primer probe set ) 是由 贾万忠 吴燕涛 闫鸿斌 李立 朱国强 李秀荣 李双男 姚刚 付宝权 于 2018-08-20 设计创作，主要内容包括：本发明公开了一种用于鉴定多房棘球绦虫引物对、引物探针组以及他们的应用。本发明提供了特异引物对,由序列2所示单链DNA分子和序列3所示单链DNA分子组成。本发明还保护引物探针组,由特异引物对和特异探针组成；特异探针自上游至下游依次由如下元件组成：区段甲、四氢呋喃和区段乙；区段甲如序列4所示,且3’末端第2位核苷酸被荧光基团修饰；区段乙如序列5所示,且5’末端第2位核苷酸被淬灭基团修饰,且3’末端进行C3 Spacer修饰。特异引物对或引物探针组可用于：鉴定多房棘球绦虫；鉴定待测样本中是否含有多房棘球绦虫。本发明提供特异引物对或引物探针组用于鉴定多房棘球绦虫,操作简便、快速、特异性强、灵敏度高。(The invention discloses a primer pair and a primer probe set for identifying Echinococcus multilocularis and application thereof. The invention provides a specific primer pair, which consists of a single-stranded DNA molecule shown in a sequence 2 and a single-stranded DNA molecule shown in a sequence 3. The invention also protects a primer probe group, which consists of a specific primer pair and a specific probe; the specific probe is composed of the following components from upstream to downstream in sequence: segment a, tetrahydrofuran, and segment b; segment A is shown as sequence 4, and the 2 nd nucleotide at the 3' terminal is modified by a fluorescent group; segment B is shown in sequence 5, and the 2 nd nucleotide at the 5 'end is modified by a quencher group, and the 3' end is modified by C3 Spacer. Specific primer pairs or primer probe sets can be used for: identifying Echinococcus multilocularis; and identifying whether the sample to be detected contains Echinococcus multilocularis. The specific primer pair or primer probe set provided by the invention is used for identifying the Echinococcus multilocularis, and has the advantages of simple and convenient operation, rapidness, strong specificity and high sensitivity.)

1. The specific primer pair consists of a single-stranded DNA molecule shown in a sequence 2 of the sequence table and a single-stranded DNA molecule shown in a sequence 3 of the sequence table.

2. The use of the specific primer pair of claim 1 in the preparation of a kit; the function of the kit is as follows (a) or (b):

(a) identifying Echinococcus multilocularis;

(b) and identifying whether the sample to be detected contains Echinococcus multilocularis.

3. A kit comprising a specific primer pair of claim 1; the function of the kit is as follows (a) or (b):

(a) identifying Echinococcus multilocularis;

(b) and identifying whether the sample to be detected contains Echinococcus multilocularis.

4. A method for identifying whether a tapeworm to be detected is Echinococcus multilocularis comprises the following steps:

using the genomic DNA of a tapeworm to be detected as a template, and adopting the specific primer pair of claim 1 to perform recombinase polymerase amplification, if effective amplification can be realized, the tapeworm to be detected is or is a candidate of Echinococcus multilocularis, and if effective amplification cannot be realized, the tapeworm to be detected is or is a candidate of Echinococcus multilocularis.

5. A method for identifying whether a sample to be detected contains Echinococcus multilocularis comprises the following steps:

using the total DNA of a sample to be detected as a template, and adopting the specific primer pair of claim 1 to perform recombinase polymerase amplification, if effective amplification can be realized, the sample to be detected contains Echinococcus multilocularis, and if effective amplification cannot be realized, the sample to be detected does not contain Echinococcus multilocularis.

6. A primer probe set consisting of the specific primer pair and the specific probe of claim 1;

the specific probe is composed of the following components from upstream to downstream in sequence: segment a, tetrahydrofuran, and segment b; segment A is shown as sequence 4 in the sequence table, and the 2 nd nucleotide counted from the 3' end is modified by a fluorescent group; segment B is shown as sequence 5 in the sequence table, and the 2 nd nucleotide counted from the 5 'end is modified by a quenching group, and the 3' end is modified by C3 Spacer.

7. Use of a primer probe set according to claim 6 for the preparation of a kit; the function of the kit is as follows (a) or (b):

(a) identifying Echinococcus multilocularis;

(b) and identifying whether the sample to be detected contains Echinococcus multilocularis.

8. A kit comprising the primer probe set of claim 6; the function of the kit is as follows (a) or (b):

(a) identifying Echinococcus multilocularis;

(b) and identifying whether the sample to be detected contains Echinococcus multilocularis.

9. A method for identifying whether a tapeworm to be detected is Echinococcus multilocularis comprises the following steps:

using the genomic DNA of a tapeworm to be detected as a template, and using the primer probe set of claim 6 to perform real-time fluorescence recombinase polymerase amplification, if effective amplification can be achieved, the tapeworm to be detected is or is a candidate of Echinococcus multilocularis, and if effective amplification cannot be achieved, the tapeworm to be detected is or is a candidate of Echinococcus multilocularis.

10. A method for identifying whether a sample to be detected contains Echinococcus multilocularis comprises the following steps:

using the total DNA of a sample to be detected as a template, and adopting the primer probe set of claim 6 to perform real-time fluorescence recombinase polymerase amplification, if effective amplification can be realized, the sample to be detected contains Echinococcus multilocularis, and if effective amplification cannot be realized, the sample to be detected does not contain Echinococcus multilocularis.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a primer pair and a primer probe set for identifying Echinococcus multilocularis and application thereof.

Background

Echinococcosis multilocularis (AE) is also called Alveolar echinococcosis, echinococcosis multilocularis, Alveolar echinococcosis and the like, and is one of the most common echinococcosis in China. It is a kind of parasitic disease of both human and animal caused by the parasitism of the middle taenia larva, Echinococcus multilocularis (the pathogen of the disease) in the lung, liver and intestine of the intermediate host, such as human and animal. The liver is the primary organ of the disease, and almost all clinical cases are liver cysts. The appearance of the vesicle formed by the echinococcus multilocularis is grape-shaped, the vesicle can almost occupy the whole liver after 1-2 years of invasive development, and then spread to other organs towards the surface of the liver, and even parasitize in the whole body cavity. The vesicular cysts ("worm cancers") grow invasively and can spread to other sites as often as malignant tumors. The disease seriously threatens the health of human beings, and if the death rate of patients is extremely high when the treatment is not carried out in time, the disease is a parasitic disease which is commonly suffered by human and animals and can cause serious consequences.

The final hosts of echinococcus multilocularis are commonly red (erythro) fox, Tibetan fox, sand fox, domestic dog and unguis canis, wolf, badger, wild cat, domestic cat and the like, and the fox and the dog are the most main infection sources. The dogs play an important role in the spreading process of the alveolar diseases, the dogs are almost domesticated in semi-farming and semi-pasturing areas in China, and the defecation areas of the dogs are very close to residential areas, so that the probability of the infection of people by worm eggs is greatly increased. The intermediate hosts of the echinococcus multilocularis are mainly rodents, common rodents in China comprise plateau voles, ulna, Chinese zokors, Qinghai voles, black-lipped rabbits and the like, and three common domestic animals (Tibetan sheep, yaks and Tibetan pigs) and the like also have reported infection with cyst larvae.

Echinococcus multilocularis adults are highly similar in appearance to Echinococcus granulosus, but are smaller in size, usually 1.2mm to 3.7mm in length, and are one of the smallest size species of the Taenia fasciata. The worm bodies mostly contain 4-5 segments, and some worm bodies containing 6 segments are found. The common components of the polypide are as follows: head, neck, juvenile, adult and pregnant. The head and neck part is roughly pear-shaped, the top of the head part comprises a top protrusion and 4 suckers, and two rings of small hooks (usually 23-36) are arranged on the top protrusion. The opening of the genital hole of the Echinococcus multilocularis is irregular, but most of the Echinococcus multilocularis is opened at the middle front part of one side of the segment, the number of testes is 16-36, the whole gestational joint is almost completely occupied by the uterus, and the uterus contains a large number of ova (about 200 ova). The intermediate-taenia (continuous-taenia) larvae of Echinococcus are called Echinococcus multilocularis, and are formed by the aggregation of numerous irregularly shaped white or chy-yellow multilocular vesicles with unclear boundaries. The vesicle develops into a spherical shape or an irregular spherical shape in a suitable intermediate host (such as a plateau vole), the size and the shape of the vesicle are very similar (the diameter is 0.1 cm-0.7 cm), the vesicle and the vesicle are mutually crosslinked and communicated, and the vesicle is wrapped by body tissues (outer membranes). The vesicle contains clear cyst fluid and metacercaria and is called a cyst; the jelly without metacercaria is called the immotile sac. In rodent bodies such as a field rat and the like, echinococcus multilocularis cysts are mostly cyst-bearing; in the unsuitable intermediate host (such as human), the vesicle is usually a vesicle group with micelle-like substances, and the vesicles contain a small amount of metacercaria (metacercaria) or even no metacercaria. Exogenous gemmation is the main asexual reproduction mode of the cyst larvae, the cyst larvae can be infected and colonized on tissues around the cyst in an invasive and transfer mode, and a few cyst larvae can also form membranes to separate new vesicles, namely the sacs, by growing inwards to form the membranes. The exogenous ascon can be separated from the focus, and can migrate to other parts through the body fluid circulation (blood and lymph fluid) to develop into new cyst larva.

Echinococcus alveolar saceus has been considered a liver carcinogen before the pathogen of Echinococcosis multicavium was completely elucidated. In 1885, a pathologist in Germany (Rudolf Virchow) found for the first time that the disease was caused by a parasite, but the parasitic disease was different from the common echinococcosis of the solitary type or cystic type, which was infectious and invasive and more dangerous. Virchow refers to the parasitosis, which is multi-compartmental (multilocular), small and numerous cysts, contains gelatinous material and a small amount of prototheca, as "echinococcosis multilocularis", which lays the foundation for echinococcus multilocularis nomenclature. It was formally established by the german scientist (Rudolf leucokart) until 1863 that it was an independent species and was named echinococcus multilocularis (e. Echinococcosis multicavium is prevalent in cold regions in a pan arctic distribution, including central europe, continental europe, the north and middle parts (extending eastward to japan) and parts of north america. The pasturing areas such as Qinghai, Ningxia, Gansu, Xinjiang, Sichuan and Tibet and the semi-farming and semi-pasturing areas are high incidence areas of the echinococcosis with multiple rooms in China, the treatment of the echinococcosis is more difficult than that of the echinococcosis with the cyst type, and partial patients need liver transplantation besides taking the focus of the operation, thereby bringing huge economic burden to the patients and families.

The RPA technique is known as recombinase polymerase amplification technique and mainly relies on three enzymes: a recombinase capable of binding single-stranded nucleic acids (oligonucleotides), a single-stranded DNA binding protein (SSB protein), a DNA polymerase with strand displacement activity. The RPA technique was established by Babraham institute, Cambridge, UK and invented by Twist Dx Biotech corporation (http:// www.twistdx.co.uk /). The RPA technology takes a nucleic acid replication mechanism of T4 bacteriophage as a principle, and the raw materials required by the reaction comprise recombinase proteins uvsX and uvsY coded by T4 bacteriophage, single-chain binding protein gp32, DNA polymerase and oligonucleotide; simultaneously, a primer, a probe, a template and ddH are required₂O and Mg²⁺And the like. The technology is based on the recombinase polymerase-mediated amplification principle, simulates DNA replication in organisms, can perform isothermal amplification on target segments at normal temperature,the method gets rid of the requirements on a thermal cycler, can quickly amplify the target fragment in a short time, and has the advantages of simplicity, convenience, rapidness, sensitivity and the like.

Therefore, the method for detecting the echinococcus RPA is simple, convenient, rapid and effective, and has important significance for preventing and controlling echinococcosis.

Disclosure of Invention

The invention aims to provide a primer pair and a primer probe set for identifying Echinococcus multilocularis and application thereof.

The invention firstly provides a specific primer pair which consists of a single-stranded DNA molecule shown in a sequence 2 of a sequence table and a single-stranded DNA molecule shown in a sequence 3 of the sequence table.

The primer pair provided by the invention is based on the RPA technology.

The invention also protects the application of the specific primer pair in the preparation of the kit; the function of the kit is as follows (a) or (b):

(a) identifying Echinococcus multilocularis;

(b) and identifying whether the sample to be detected contains Echinococcus multilocularis.

The invention also provides a kit, which comprises the specific primer pair; the function of the kit is as follows (a) or (b):

(a) identifying Echinococcus multilocularis;

(b) and identifying whether the sample to be detected contains Echinococcus multilocularis.

The invention also provides a method for identifying whether the tapeworm to be detected is Echinococcus multilocularis, which comprises the following steps:

and (3) using the genomic DNA of the tapeworm to be detected as a template, and adopting the specific primer pair to perform recombinase polymerase amplification, wherein if effective amplification can be realized, the tapeworm to be detected is or is a candidate of the Echinococcus multilocularis, and if effective amplification cannot be realized, the tapeworm to be detected is or is a candidate of the Echinococcus multilocularis.

The invention also provides a method for identifying whether the sample to be detected contains Echinococcus multilocularis, which comprises the following steps:

and (3) performing recombinase polymerase amplification by using the total DNA of the sample to be detected as a template and adopting the specific primer pair, wherein if the effective amplification can be realized and the sample to be detected contains the Echinococcus multilocularis, if the effective amplification cannot be realized, the sample to be detected does not contain the Echinococcus multilocularis.

In the method, the judgment is realized by electrophoresis detection.

The method is used for non-diagnostic purposes.

The invention also protects a primer probe group, which consists of the specific primer pair and the specific probe;

the specific probe is composed of the following components from upstream to downstream in sequence: segment a, tetrahydrofuran, and segment b; segment A is shown as sequence 4 in the sequence table, and the 2 nd nucleotide counted from the 3' end is modified by a fluorescent group; segment B is shown as sequence 5 in the sequence table, and the 2 nd nucleotide counted from the 5 'end is modified by a quenching group, and the 3' end is modified by C3 Spacer.

The primer probe set provided by the invention is based on the RPA technology.

The fluorophore may specifically be FAM. The quencher group may specifically be BHQ 1.

The invention also protects the application of the primer probe group in the preparation of a kit; the function of the kit is as follows (a) or (b):

(a) identifying Echinococcus multilocularis;

(b) and identifying whether the sample to be detected contains Echinococcus multilocularis.

The invention also provides a kit, which comprises the primer probe group; the function of the kit is as follows (a) or (b):

(a) identifying Echinococcus multilocularis;

(b) and identifying whether the sample to be detected contains Echinococcus multilocularis.

The invention also provides a method for identifying whether the tapeworm to be detected is Echinococcus multilocularis, which comprises the following steps:

and (3) performing real-time fluorescence recombinase polymerase amplification by using the genome DNA of the tapeworm to be detected as a template and adopting the primer probe set, wherein if effective amplification can be realized, the tapeworm to be detected is or is a candidate of the Echinococcus multilocularis, and if the effective amplification cannot be realized, the tapeworm to be detected is or is a candidate of the Echinococcus multilocularis.

The invention also provides a method for identifying whether the sample to be detected contains Echinococcus multilocularis, which comprises the following steps:

and (3) performing real-time fluorescence recombinase polymerase amplification by using the total DNA of the sample to be detected as a template, wherein if the effective amplification can be realized and the sample to be detected contains the Echinococcus multilocularis, and if the effective amplification cannot be realized and the sample to be detected does not contain the Echinococcus multilocularis.

In the method, the judgment is specifically realized by detecting fluorescence.

The method is used for non-diagnostic purposes.

Any one of the above tapeworms to be detected is taenia multiceps, taenia buqueta, taenia sojae, echinococcus multilocularis, echinococcus granulosus or echinococcus shikoshii.

Any one of the above samples to be tested is a sample from a host animal of Echinococcus multilocularis.

The primer probe set provided by the invention is used for detecting the Echinococcus multilocularis by using the RPA technology, and the monomolecular nucleic acid detection can be carried out within 15 minutes at normal temperature. The specific primer pair or primer probe set provided by the invention is used for identifying Echinococcus multilocularis, is simple and convenient to operate, is rapid, has strong specificity and high sensitivity, and is particularly suitable for the fields of in-vitro diagnosis, veterinarian, food safety, biological safety, agriculture and the like.

Drawings

FIG. 1 is an electrophoresis diagram of PCR amplification using specific primer pairs in example 1.

FIG. 2 is an electrophoretogram of PCR amplification of formazan using control primers in example 1.

FIG. 3 is an electrophoretogram of PCR amplification of B using control primers in example 1.

FIG. 4 is an electrophoretogram of RPA performed using specific primer pairs in example 1.

FIG. 5 shows the results of example 2.

FIG. 6 shows the result of step two in example 3.

FIG. 7 shows the results of step three in example 3.

FIG. 8 shows the result of step one in example 4.

FIG. 9 shows the results of step two in example 4.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.