NL2038125B1

NL2038125B1 - crRNA AND KIT FOR TESTING ECHINOCOCCI ON BASIS OF RAA COMBINED WITH CRISPR-Cas12a, AND APPLICATION OF crRNA

Info

Publication number: NL2038125B1
Application number: NL2038125A
Authority: NL
Inventors: Zhang Huan; Li Li; Yan Hongbin; Wang Meng; Zhang Nianzhang; Qu Zigang; Fu Baoquan; Li Wenhui
Original assignee: Lanzhou Veterinary Res Institute Chinese Academy Of Agricultural Sciences Lanzhou Branch Center Of C
Priority date: 2024-01-25
Filing date: 2024-07-03
Publication date: 2025-08-06
Also published as: CN118006605A

Abstract

The present invention provides a clustered regularly interspaced short palindromic repeat ribonucleic acid (chNA) and kit for testing echinococci on the basis of recombinase aided amplification (RAA) combined with CRISPR—Calea, and application of the chNA, which belong to the technical field of medical testing. The chNA for testing echinococci on the basis of RAA combined with CRISPR—Calea has a nucleotide sequence shown as SEQ ID NO:l. The present invention further provides a kit for testing. The kit includes the chNA, Calea, a singlestranded DNA—fluorophore—quencher (ssDNA—FQ) and RAA primers. The kit provided by the present invention has good testing specificity and relatively high sensitivity, has obvious advantages compared with a polymerase chain reaction (PCR) amplification method, and provides a new means for clinical testing of infection with echinococcus granulosus and echinococcus multilocularis.

Description

CrRNA AND KIT FOR TESTING ECHINOCOCCI ON BASIS OF RAA

COMBINED WITH CRISPR-Casl2a, AND APPLICATION OF crRNA

TECHNICAL FIELD

[01] The present invention belongs to the technical field of medical testing, and particularly relates to a clustered regularly interspaced short palindromic repeat ribonucleic acid (crRNA) and kit for testing echinococci on the basis of recombinase aided amplification (RAA) combined with CRISPR-CaslZa, and application of the crRNA.

BACKGROUND ART

[02] Echinococcus multilocularis (Em) is similar to echinococcus granulosus (Eg) in morphology and structure, but the adult of echinococcus multilocularis is smaller, only 1.2-3.7 mm long, averaging 2.13 mm. The scolex, apical protrusion, hook and sucker are correspondingly smaller, and 13-34 hooks grow on the apical protrusion.

There are usually 4-5 proglottides in the worm body.

Genital pores are located in front of the midline of the proglottis, and the number of testes is relatively small, ranging from 26 to 36, all of which are located behind the genital pores. The uterus of the gravid proglottis is in a simple cyst shape without lateral sac, containing 187-404 eggs. The egg shape and size are difficult to distinguish from those of echinococcus granulosus. In addition, the life cycles of echinococcus multilocularis and echinococcus granulosus are similar. The common final host of echinococcus multilocularis is fox, followed by dog, wolf, badger, cat, etc. Echinococcus granulosus can also be parasitized in the final host of echinococcus multilocularis. Echinococcus multilocularis and echinococcus granulosus can infect not only animals but also humans. Clinical manifestations are that the infection destroys liver tissue especially seriously, which can cause liver failure and lead to liver coma, or induce liver «cirrhosis and cause portal hypertension, complicated with massive hemorrhage of gastrointestinal tract and even death. Therefore, whether to be infected with echinococcosis or not is clinically determined by simultaneously testing echinococcus multilocularis and echinococcus granulosus.

[03] At present, diagnostic methods for echinococcosis granulosa and alveolar echinococcosis usually include X- ray, B-ultrasound, CT, isotope scanning and various immunological tests. However, the existing diagnostic methods have the problem of complicated testing operation.

With the maturity of molecular testing technology, the polymerase chain reaction (PCR) amplification technology has been developed to diagnose infection with Eg and Em.

However, the testing sensitivity of this method is low and fails to satisfy the requirements of clinical testing.

SUMMARY

[04] In view of this, an objective of the present invention is to provide a clustered regularly interspaced short palindromic repeat ribonucleic acid (crRNA) for testing echinococci on the basis of recombinase aided amplification (RAA) combined with CRISPR-CaslZa. The crRNA has the ability to highly specifically bind genes of echinococcus granulosus (Eg) and echinococcus multilocularis (Em), which ensures the high testing sensitivity of Eg and Em.

[05] The present invention provides a crRNA for testing echinococci on the basis of RAA combined with CRISPR-

Casl2a, which has a nucleotide sequence shown as SEQ ID

NO:1.

[06] The present invention provides a kit for testing echinococci on the basis of RAA combined with CRISPR-

Casl2a, which includes the crRNA, Casl2a, a single- stranded DNA-fluorophore-quencher (ssDNA-FQ) and RAA primers.

[07] Preferably, the RAA primers include a forward primer having a nucleotide sequence shown as SEQ ID NO:2 and a reverse primer having a nucleotide sequence shown as SEQ

ID NO:3.

[08] Preferably, a nucleotide sequence of the ssDNA-FQ is shown as a fluorophore-TTATT-quencher group.

[09] Preferably, the kit further includes positive plasmids.

[10] The positive plasmids include a recombinant plasmid containing a 18s rRNA sequence of echinococcus multilocularis and a recombinant plasmid containing a 18s rRNA sequence of echinococcus granulosus.

[11] Preferably, amplification primers for the 18s rRNA sequence of echinococcus multilocularis or the 18s rRNA sequence of echinococcus granulosus include a forward primer having a nucleotide sequence shown as SEQ ID NO:4 and a reverse primer having a nucleotide sequence shown as

SEQ ID NO:5.

[12] The present invention provides application of the

CrRNA combined with Casl2a, an ssDNA-FQ and RAA primers in preparation of a kit for testing echinococci. The RAA primers include a forward primer having a nucleotide sequence shown as SEQ ID NO:2 and a reverse primer having a nucleotide sequence shown as SEQ ID NO:3, and the echinococci include echinococcus multilocularis and/or echinococcus granulosus.

[13] The present invention provides a method for testing echinococcus multilocularis and/or echinococcus granulosus for non-disease diagnostic purposes. The method includes the following steps:

[14] employing an RAA technology to amplify the 185 rRNA sequence of a sample to be tested to obtain amplification products; and

[15] mixing the amplification products with the crRNA,

Casl2a and an ssDNA-FQ to perform a CRISPR-Casl12a reaction, testing a fluorescence signal, and determining whether infection with echinococcus multilocularis and/or echinococcus granulosus occurs in the tested sample according to whether the fluorescence signal exists or not.

[16] Preferably, a reaction is performed at 39°C for 15- 25 min during amplification by using the RAA technology.

[17] Preferably, during the CRISPR-Casl2a reaction, a working concentration of the crRNA is 100-800 uM, a working concentration of the Casl2a is 400-800 uM, and a working concentration of the ssDNA-FQ is 750-850 uM.

[18] The CRISPR-Casl2a reaction is performed at 37°C for 15-25 min.

[19] The present invention provides a crRNA for testing echinococci on the basis of RAA combined with CRISPR-

Casl2a, which has a nucleotide sequence shown as SEQ ID

NO:1. According to the present invention, common conserved regions of 18S rRNA genes in echinococcus multilocularis and echinococcus granulosus are used as templates to screen an appropriate PAM (5(-TTTN-3{) sequence, which relates to a series of crRNAs. By means of testing and screening with the RAA combined with CRISPR-Casl2a, it is found that the crRNA shown in SEQ ID NO:1 has the highest fluorescence value, which indicates that the crRNA has the strongest binding property with the template DNA, thereby providing guarantee for high sensitivity and high-accuracy testing of the echinococci.

[20] The present invention further provides a method for testing echinococcus multilocularis and/or echinococcus granulosus for non-disease diagnostic purposes. An RAA technology is employed to amplify the 18S rRNA sequence of a sample to be tested to obtain amplification products.

The amplification products are mixed with the crRNA,

Casl2a and an ssDNA-FQ to perform a CRISPR-Casl2a reaction, a fluorescence signal is tested, and whether infection with echinococcus multilocularis and/or echinococcus granulosus occurs in the tested sample is determined according to whether the fluorescence signal exists or not.

According to the present invention, the RAA technology and the CRISPR-Casl2a testing technology are combined, such that testing sensitivity is greatly improved. A testing minimum limit of the Em DNA is 0.1 pg/uL, and a testing minimum limit of the Eg DNA is 0.1 pg/uL. Compared with a polymerase chain reaction (PCR) testing method, this 5 method has great advantages. Moreover, the specificity testing results show that the method only realizes testing of echinococcus multilocularis and/or echinococcus granulosus, and cannot realize testing of other echinococci or parasites of other species, which indicates that the method provided by the present invention has good testing specificity.

BRIEF DESCRIPTION OF THE DRAWINGS

[21] FIG. 1 shows results of fluorescence values of different kinds of crRNAs after a CRISPR-Casl2a reaction;

[22] FIG. 2 shows results of RAA {fluorescence values under different reaction time;

[23] FIG. 3 shows results of fluorescence values of a

CrRNA and Casl2a at different concentration combinations;

[24] FIG. 4 shows results of fluorescence values under different CRISPR-CaslZa reaction time;

[25] FIG. 5 shows specificity testing results during testing of echinococcus granulosus and echinococcus multilocularis by using a method of combining RAA with

CRISPR-CaslZa, where graph A shows results of fluorescence values of CRISPR-Casl2a testing, picture B shows photos of reaction tubes after blue light irradiation, and tubes 1-8 are toxoplams gondii, taenia multiceps, T. hydatigena, hydatigen taeniaeformis, trichinella spiralis, echinococcus shiquicus, fasciola hepatica, and F. gigantica respectively;

[26] FIG. 6 shows sensitivity testing results for different concentrations of Em genomic DNAs in Example 7 of the present invention, where graph A shows fluorescence value results of CRISPR-CaslZa testing, the sensitivity is 0.1 pg/uL, picture B shows results of CRISPR-CaslZa testing reaction tubes after blue light irradiation, tubes

1-5 are different final DNA concentrations, which are 4, 1, 0.2, 0.1 and 0.02 pg/uL respectively, and tube 6 is a negative control;

[27] FIG. 7 shows sensitivity testing results for different concentrations of Eg genomic DNAs in Example 7 of the present invention, where graph A shows fluorescence value results of CRISPR-Casl2a testing, the sensitivity is 0.1 pg/uL, picture B shows results of CRISPR-Casl2a testing reaction tubes after blue light irradiation, tubes 1-5 are different final DNA concentrations, which are 4, 1, 0.2, 0.1 and 0.02 pg/uL respectively, and tube 6 is a negative control;

[28] FIG. 8 shows PCR amplification results of different concentrations of Em and Eg genomic DNAs in Comparative

Example 1 of the present invention, where 1-5 are Em, 7-11 are Eg, the final DNA concentrations are 10, 2.5, 0.5, 0.25 and 0.05 pg/uL respectively, and 6 and 12 are negative controls, which shows that the sensitivity of Em is 2.5 pg/uL, and the sensitivity of Eg is 0.25 pg/uL; and

[29] FIG. 9 shows results of fecal testing for dogs infected with Eg under different days.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[30] The present invention provides a clustered regularly interspaced short palindromic repeat ribonucleic acid (CrRNA) for testing echinococci on the basis of recombinase aided amplification (RAA) combined with

CRISPR-Casl2a, which has the nucleotide sequence shown as

SEQ ID NO:1 (UAAUUUCUACUAAGUGUAGAUUUGCGGUAGGGUGGUUGGUUC) .

[31] The present invention provides a kit for testing echinococci on the basis of RAA combined with CRISPR-

Casl2a, which includes the crRNA, Casl2a, a single- stranded DNA-fluorophore-quencher (ssDNA-F9Q) and RAA primers.

[32] In the present invention, the RAA primers preferably include a forward primer having a nucleotide sequence shown as SEQ ID NO:2 (GCCTGCTAATTAGTGCGTTCGTCCACTGCGC) and

: a reverse primer having a nucleotide sequence shown as SEQ

ID NO:3 (CTGTTATTGCTCTATCTCGCGCGGCTTCTCC). The RAA primers are designed and obtained according to common conserved regions in 18s rRNA sequences in echinococcus multilocularis and echinococcus granulosus.

[33] In the present invention, a nucleotide sequence of the ssDNA-FQ is shown as a fluorophore-TTATT-quencher group. The present invention has no special limitation on the type of the fluorescent group or quenching group, and fluorescent groups or quenching groups well known in the art may be used. In the examples of the present invention, the fluorescent group preferably includes 6-FAM, and the quenching group preferably includes BHQL.

[34] In the present invention, preferably, the kit further includes two positive plasmids. The two positive plasmids include a recombinant plasmid containing a 18s rRNA sequence of echinococcus multilocularis and a recombinant plasmid containing a 18s rRNA sequence of echinococcus granulosus. Amplification primers for the 18s rRNA sequence of echinococcus multilocularis or the 18s rRNA sequence of echinococcus granulosus include a forward primer having a nucleotide sequence shown as SEQ ID NO:4 and a reverse primer having a nucleotide sequence shown as

SEQ ID NO:5. The present invention has no special limitation on the skeleton plasmid of the positive plasmid, and skeleton plasmids well known in the art may be used, such as a pMD-18T vector. The present invention has no special limitation on a construction method for the positive plasmid, and a recombinant plasmid construction method well known in the art may be employed. Sequencing verification is performed after the positive plasmid is constructed, and the recombinant plasmid containing the target gene segment is obtained as a positive plasmid.

[35] In the present invention, the testing principle of the kit is to amplify the sample or positive plasmid with

RAA primers, mix the obtained amplified fragment with the crRNA, the Casl2a and the ssDNA-FQ, combine the Casl2a and the crRNA to form a Casl2a-crRNA complex first, then recognize a prespacer adjacent motif (PAM) at the target

DNA, complementarily combine cCrRNA with the target strand in the DNA double strand to form a loop, unwind the DNA double strand, and activate the conformation of the endonuclease catalytic site. Since this endonuclease site can only be inserted into one DNA strand at a time, the target DNA double strand breaks sequentially, the non- target strand is sheared first, and then the target strand is sheared. After the cleavage product is released from the complex, the ruvc endonuclease catalytic site of

Casl2a protein remains active, and arbitrary single- stranded DNA can be sheared and degraded randomly. Based on this principle, the single-stranded DNA is prepared into a fluorescence quenching probe, and specificity testing of the target sequences is realized by monitoring the release of fluorescence signals.

[36] The present invention provides application of the

CrRNA combined with CaslZa, an ssDNA-FQ and RAA primers in preparation of a kit for testing echinococci. The RAA primers include a forward primer having a nucleotide sequence shown as SEQ ID NO: 2 (GCCTGCTAATTAGTGCGTTCGTCCACTGCGC) and a reverse primer having a nucleotide sequence shown as SEQ ID NO:3 (CTGTTATTGCTCTATCTCGCGCGGCTTCTCC), and the echinococci include echinococcus multilocularis and/or echinococcus granulosus.

[37] The present invention provides a method for testing echinococcus multilocularis and/or echinococcus granulosus for non-disease diagnostic purposes. The method includes the following steps:

[38] employ an RAA technology to amplify the 183 rRNA sequence of a sample to be tested to obtain amplification products; and

[39] mix the amplification products with the crRNA,

Casl2a and an ssDNA-FQ to perform a CRISPR-Casl2a reaction, test a fluorescence signal, and determine whether infection with echinococcus multilocularis and/or echinococcus granulosus occurs in the tested sample according to whether the fluorescence signal exists or not.

[40] In the present invention, a reaction is preferably performed at 39°C for 15-25 min during amplification by using the RAA technology, and reaction time is more preferably 15 min. An RAA reaction time optimization experiment shows that during the reaction time of 5-35 min, the fluorescence value generated under the reaction time of 15-25 min is relatively high, and the fluorescence value under the reaction time of 15 min is the highest.

Moreover, the reaction time is relatively short, and therefore, the optimal reaction time of RAA is 15 min.

[41] In the present invention, during the CRISPR-Casl12a reaction, a working concentration of the crRNA is preferably 100-800 uM, a working concentration of the

CaslZa is preferably 400-800 UM, and a working concentration of the ssDNA-FQ is preferably 750-850 pM. In the examples of the present invention, to provide better testing results, use concentrations of the CaslZa and

CrRNA in the CRISPR-Casl2a reaction system are optimized, and the concentrations are both within the range of 100- 800 nM. When the concentrations of the crRNA and CaslZa are 100 nM:800 nM, 400 nM:400 nM, and 800 nM:600 nM, the fluorescence value is relatively high. In view of the testing cost, the group with the lower Casl2a concentration is selected, that is the reaction system with the crRNA:CaslZa concentration being 400:400 nM.

[42] In the present invention, the CRISPR-Casl2a reaction is preferably performed at 37°C for 15-25 min. Optimization experiment results of the CRISPR-Casl2a reaction time show that the fluorescence value will be increased with the reaction time within the time of 10-50 min. In order to control the reaction time of the whole experiment, 20 min is selected as the reaction time of the CRISPR-Casl2a.

[43] In the present invention, specificity tests are performed by using the above method. Results show that echinococcus multilocularis, echinococcus granulosus, echinococcus shiquicus, taenia multiceps, T. hydatigena, hydatigen taeniaeformis, fasciola hepatica, F. gigantica, trichinella spiralis and toxoplams gondii are test objects, only echinococcus multilocularis and echinococcus granulosus emit visible fluorescence under blue light irradiation, and fluorescence is not observed in other test objects. Fluorescence values tested by the CRISPR-

Casl2a testing system show that the fluorescence values of echinococcus multilocularis and echinococcus granulosus are higher and significantly different from other test objects. The method provided by the present invention has the characteristic of strong specificity.

[44] In the present invention, sensitivity testing experiments are performed by using the above method, and results show that the minimum test limit of echinococcus multilocularis is 0.1 pg/uL, and the minimum test limit of echinococcus granulosus is 0.1 pg/uL. The test limit of polymerase chain reaction (PCR) is 2.5 pg/uL for echinococcus multilocularis and 0.25 pg/uL for echinococcus granulosus, which indicates that sensitivity of the RAA combined with CRISPR-Casl12a is higher than that of PCR testing {for echinococcus multilocularis and echinococcus granulosus.

[45] The crRNA and kit for testing echinococci on the basis of RAA combined with CRISPR-Casl2a, and application of the crRNA provided by the present invention are described in detail below in conjunction with the examples, but they are not to be construed as limiting the protection scope of the present invention.

[46] Description of sources of materials, instruments and reagents

[47] 1 Biological materials

[48] DH5a receptor cells, echinococcus granulosus (Eg), echinococcus multilocularis (Em), toxoplams gondii, taenia multiceps, T. hydatigena, hydatigen taeniaeformis,

trichinella spiralis, echinococcus shiquicus, fasciola hepatica, and F. gigantica.

[49] 2 Main reagents

[50] Premix Tag™ DNA polymerase and pMD18-T vector kits were purchased from Takara Biomedical Technology (Beijing)

Co., Ltd., universal DNA purification and recycling kits and blood/cell/tissue genomic DNA extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd., and plasmid extraction kits were purchased from Omega Bio-Tek

Company. RAA nucleic acid amplification reagents (basic type) were purchased from Hangzhou ZC Bio-Sci&Tech Co.

Ltd., Nuclease-Free Water was purchased from QIAGEN

Company, and 10xNE Buffer and EnGen Lba Casl2a were purchased from NEB (USA) Company.

[51] 3 Instruments

[52] A PCR instrument and a qPCR instrument were purchased from BIO RAD (USA) company, a small desktop centrifuge was purchased from Eppendorf (Germany) company, and a gel imaging system and a dry constant temperature metal bath as well as a gel cutter, etc. were from Tiangen

Biotech (Beijing) Co., Ltd.

[53] Example 1

[54] Design of primers and construction of positive plasmids

[55] Mitochondrial 18S rRNA gene sequences of Em (GenBank:AB731634.1) and Eg (GenBank:G0260092.1) were downloaded from the National Center of Biotechnology

Information (NCRI} website. After sequence alignment by using MEGA 7 software, primers were designed by using

Primer Premier 5 software for Em and Eg conserved regions.

The upstream sequence of the universal primer was 5-

TGTTGGTGGGCCGGTCAGTAT-3 (SEQ ID NO:4), and the downstream sequence was b-CGGCTTCTCCCGCTTGTCCCTC-3 (SEQ ID NO:5), which were synthesized by Sangon Biotech (Shanghai) Co.,

Ltd.

[56] DNAs of Eg and Em were extracted respectively, and by using the DNAs of Eg and Em as templates, PCR amplification was performed. Then, amplification products were purified and inserted into an EcoRV multiple cloning site of the pMD-18T vector to construct recombinant plasmids. The recombinant plasmids were sent to Beijing

Tsingke Biotech Co., Ltd. for sequencing. Sequence alignment with the 18S DNA gene sequence by using the MEGA 7 software showed that when sequencing results were consistent with the expected 185 rRNA, and the recombinant vectors were determined as positive recombinant plasmids.

[57] Example 2

[58] Design and screening method for crRNA

[59] After aligning the 18S rRNA genes of Eg and Em, the appropriate PAM (5-TTTN-3) sequence was screened, and then the corresponding crRNA was designed as shown in Table 1, which was specifically synthesized by Shanghai GenePharma

Co., Ltd. An ssDNA-FQ report probe was synthesized by

Sangon Biotech (Shanghai) Co., Ltd.

[60] Table 1 Sequence information of crRNA and ssDNA-FQ

Name Sequence (5-3)

CrERNAT UAAUUUCUACUAAGUGUAGAUCGCUCUUGCCCUCUCCUCUCE (SEQ ID NO:6) — UAAUUUCUACUAAGUGUAGAUCUCGCUCGUUCGCUCUCUUGC (SEQ ID NO:7)

CTRNA3 UAAUUUCUACUAAGUGUAGAUUUGCGGUAGGGUGGUUGGUUC {SEQ ID NO:1)

CrERNAA VAAUUUCUACUAAGUGUAGAUGCGUUGGCCCGCGGGUGCGGE (SEQ ID NO:8) ssDNA-FQ 5'-6-FAM-TTATT-3'BHQ1

[61] The four synthesized crRNA primers were screened. 10 uL of each of crRNA4, Caslza and ssDNA-FQ with the concentration of 800 nM was added to an eight-connected tube of 200 uL, and finally, 2 uL of positive plasmid was added. After uniform mixing, the FAM fluorescence value was measured after a thermostatic reaction for 49 min at 37°C in a metal bath.

[62] The recombinant plasmids of Eg and Em were used as target DNAs to screen the designed crRNAs.

[63] Results are shown in FIG. 1. The crRNA-3 has higher fluorescence values than other crRNAs, so the crRNA-3 was selected for subsequent experiments.

[64] Example 3

[65] Design of RAA primers and RAA method

[66] RAA primers capable of amplifying the screened crRNA were designed in the conserved regions of 18S rRNA sequences of Eg and Em, and synthesized by Sangon Biotech {Shanghai} Co., Ltd.

[67] Table 1 Sequence information of RAA primers

Name of

Sequence (b5'-3") primer

S-F GCCTGCTAATTAGTGCGTTCGTCCACTGCGC (SEQ ID NO:2)

S-R CTGTTATTGCTCTATCTCGCGCGGCTTCTCC (SEQ ID NO:3)

[68] The RAA reaction system was composed of 25 uL of A

Buffer, 17.5 1uL of ddH:0, 2 pL of each of upstream and downstream primers, 2.5 uL of B Buffer, and 1 uL of the

DNA template. RAA was performed after uniform mixing of the required reagents. Specifically, a thermostatic reaction was performed at 39°C for 30 min in a metal bath, 50 uL of phenol: chloroform: iscamyl alcohol (volume ratio of 25:24:1) extract was added, and the mixture was mixed uniformly. Then, centrifugation was performed at 12000 rpm/min for 5 min, and supernatant was taken for electrophoresis or CRISPR-Casl2a testing.

[69] Example 4

[70] Optimization experiment of RAA reaction time

[71] According to the RAA method of Example 3, reaction time gradients of 5, 10, 15, 20, 25, 30, and 35 minutes at a constant temperature of 39°C were set for RAA reaction time, and the optimal reaction time was screened.

[72] For RAA reaction with different reaction time, fluorescence results were shown in FIG. 2. The fluorescence value under the time of 15-25min was higher than that under other reaction time, the fluorescence value under the time of 15 min was the highest, and the reaction time was relatively short, such that RAA reaction time was determined to be 15 min.

[73] Example 5

[74] Optimization of CRISPR-Casl2a reaction system

[75] RAA was performed by using positive plasmids containing 18S rRNA sequences of Eg and Em as templates respectively, and RAA was specifically performed according to the optimization time screened in Example 4. After amplification products were obtained, the CRISPR-Casl2a reaction system was prepared, and the CRISPR-CaslZa reaction was performed at 37°C for 49 min.

[76] CRISPR-CaslZa reaction system was prepared, which was specifically composed of 10 uL of each of a crRNA,

Casl2a and an ssDNA-FQ (800 nM), and 2 uL of positive recombinant plasmid. In order to find the optimal reaction system for CRISPR-CaslZa testing, the concentrations of

CrRNA and Casl2a were optimized. The crRNA and CaslZa were diluted to 100 nM, 200 nM, 400 nM, 600 nM and 800 nM in sequence, and fluorescence values were tested after different concentrations were arranged and combined.

[77] As shown in FIG. 3, when the concentrations of the

CrRNA and the Casl2a were 100:800, 400:400 and 800:600 nM, the fluorescence value was relatively high. In view of control over the testing cost, the group with the lower concentration of Casl2a was selected, that is, the reaction system with crRNA: Casl2a concentration being 400 nM:400 nM.

[78] Example 6

[79] Optimization of CRISPR-Casl2a reaction system

[80] RAA was performed by using positive plasmids containing 18S rRNA sequences of Eg and Em as templates respectively, and RAA was specifically performed according to the optimization time screened in Example 4. After amplification products were obtained, the CRISPR-Casl2a reaction system was prepared, which was specifically composed of 10 uL of each of the crRNA (400 nM), Casl2a (400 nM), and the ssDNA-FQ (800 nM), and 2 uL of positive recombinant plasmid. The reagents and plasmids required for the CRISPR-Casl2a reaction were mixed uniformly and put into a qPCR instrument. The thermal cycle temperature was set at 37°C, plate reading was performed once every 2 minutes for 26 cycles in total, and the change of fluorescence values was observed. Reasonable reaction time was selected.

[81] As shown in FIG. 4, the fluorescence value would be increased with reaction time. In order to control the overall test time, 20 min was chosen as the reaction time for CRISPR-Casl2a.

[82] Example 7

[83] Specificity test

[84] RAA was sequentially performed by taking DNAs of echinococcus multilocularis, echinococcus granulosus, echinococcus shiquicus, taenia multiceps, T. hydatigena, hydatigen taeniaeformis, fasciola hepatica, F. gigantica, trichinella spiralis and toxoplams gondii as templates respectively. The specific method was to prepare an RAA reaction system which was composed of 25 uL of A Buffer, 17.5 UL of ddH;0, 2 |L of each of upstream and downstream primers, 2.5 uL of B Buffer, and 1 uL of DNA template. RAA was performed after required reagents were uniformly mixed, and specifically, a thermostatic reaction was performed at 39°C for 15 min in a metal bath. Fluorescence values of amplification products were tested by using a

CRISPR-CaslZa system. Specifically, the CRISPR-Casl2a reaction system was prepared, which was composed of 10 uL of each of the crRNA (400 nM), the Casl2a (400 nM) and the

SsDNA-FQ (800 nM), and 2 uL of the amplification product.

The CRISPR-Casl2a reaction system was put into a gPCR instrument. The thermal cycle temperature was set at 37°, and time was set as 20 min. The change of fluorescence values was observed.

[85] The fluorescence values were tested by CRISPR-Casl2a4, and results were shown in graph A of FIG. 5. Except for Eg and Em which had relatively high fluorescence values,

others were similar to those of the negative control. Blue light irrigation reaction tubes were photographed. Results are shown in picture B of FIG. 5, and only Eg and Em emits visible fluorescence, indicating that the testing method provided by the present invention has good testing specificity and can only test echinococcus multilocularis and echinococcus granulosus.

[86] Example 8

[87] Sensitivity test

[88] Final concentrations of Eg and Em genomic DNAs were diluted to 4 pg/uL, 1 pg/uL, 0.2 pg/uL, 0.1 pg/uL and 0.02 pg/uL in seguence, and RAA was performed respectively.

Fluorescence values were tested by using the CRISPR-Casl2a system.

[89] The fluorescence values of DNA amplification at different concentrations were tested by using the CRISPR-

Casl2a, and results were shown in graph A of FIG. 6 and graph A of FIG. 7. The minimum test limit of the Em DNA was 0.1 pg/uL, and the minimum test limit of the Eg DNA was 0.1 pg/uL. The reaction tubes were observed under blue light, and the fluorescence showed a trend from strong to weak as shown in picture B of FIG. 6 and picture B of FIG. 7.

[90] Comparative Example 1

[91] Test of testing sensitivity of Eg and Em by using

PCR method

[92] PCR primers (Zhu GQ, Yan HB, Li L, et al. First report on the phylogenetic relationship, genetic variation of Echinococcus shiguicus isolates in Tibet Autonomous

Region, China. Parasit Vectors. 2020;13(1) :590. Published 2020 Nov 23. Doi:10.1186/s13071-020-04456-w) capable of amplifying Em and Eg were designed in conserved regions of coxl sequences of Eg and Em, and synthesized by Sangon

Biotech (Shanghai) Co., Ltd.

[93] Table 3 Sequence information of PCR primers

Name of

Sequence (5 (-3 {() primer cox1l-F AGTTACTGCTAATAATTTTGTGTCAT (SEQ ID NO:9) cox1l-R ATGATGTAAAAGGCAAATAAACC (SEQ ID NO:10)

[94] Notes: primers in Table 3 are from the literature of

First report on the phylogenetic relationship, genetic variation of Echinococcus shiquicus isolates in Tibet

Autonomous Region, China.

[95] Final concentrations of Eg and Em genomic DNAs were diluted to 10, 2.5, 0.5, 0.25 and 0.05 pg/ul in sequence, and PCR amplification was performed respectively.

Specifically, a PCR reaction system was prepared, including 10 uL of Ex Tag Premix, 1 uL of each of upstream and downstream primers, 7 uL pf ddH:0 and 1 pL of DNA template. The required reagents were mixed uniformly, and then, PCR amplification was performed. Specifically, predenaturation was performed at 95°C for 5 min, denaturation was performed at 95°C for 30 s, annealing was performed at 61°C for 30 s, and extension was performed at 72°C for 2 min with 35 cycles in total. Final extension was performed at 72°C for 10 min. Amplification products obtained were subjected to agarose gel electrophoresis, and results were checked and compared with the results of the sensitivity test.

[96] PCR amplification was performed by taking genomic

DNAs of Em and Eg at different concentrations as templates. Electrophoresis results are shown in FIG. 8.

The minimum test limit of Em is 2.5 pg/uL, and the minimum test limit of Eg is 0.25 pg/ul, indicating that sensitivity during testing of Em and Eg by using RAA combined with CRISPR-Casl2a is higher than that of PCR testing.

[97] Example 9

[98] Method for testing Eg and Em on basis of RAA combined with CRISPR-CaslZa

[99] Feces of dogs infected with Eg for different days were collected, and fecal DNAs were extracted. RAA and

CRISPR-Casl2a testing were performed by using the extracted fecal DNAs as templates according to the method of Example 7, and results were recorded. Fecal samples were collected from various regions, fecal DNAs were extracted, testing on the basis of RAA combined with

CRISPR-Casl2a was performed, and results were recorded.

[100] Table 4 Testing results of fecal samples collected from various regions

Number of Em, Eg Microscopic

Em, Eg

Category Name samples (PCR) examination (sample) (sample) (sample) (sample)

Region Maqu 49 7 1 16

Jingyuan 65 26 27 42

Sex Female 4 1 2 1

Male 70 18 25 37

Unknown 40 14 1 20

Age <1 year 7 3 3 3 2-4 years 50 10 21 24 z 4 years 17 6 3 10

Unknown 40 14 1 19

Type Fox 24 7 1 16

Dog 90 26 27 42

Total 114 33 28 58 number

[101] As shown in Table 4, 33 positive samples were detected in the 114 samples tested by using the method of combining RAA with CRISPR-Casl12a, and 28 positive samples were detected by using the PCR method. Em and Eg could not be distinguished by means of microscopic examination, and 58 samples containing eggs were detected.

[102] FIG. 9 shows results of fecal testing for dogs infected with Eg under different days, indicating that positive results can be detected between 16 and 70 days of infection.

[103] The above mentioned descriptions are merely the preferred embodiments of the present invention, it should be pointed out that those of ordinary skill in the art can also make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also fall within the protection scope of the present invention.

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Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences(Lanzhou Branch

Center of China Animal Health And Epidemiology Center) </ApplicantName> —invention Title languageCode="en"> crRNA AND KIT FOR TESTING ECHINOCOCCI ON BASIS OF RAA COMBINED WITH

CRISPR-Cas12a, AND APPLICATION OF crRNA </InventionTitle> <SeguenceTotalQuantity>10</Sequence VotalQuantity> —<Seguencebata sequenceIDNumber="1"> —<INSDSeg> <INSDSeg lengih>42</INSDSeqg length> <INSDSeg moltyype>RNAJINSDSeg wmoltype> <INSDseg division>PATZENSBSeqg division> —NSDseg Testure-{abie> —<INSDFesture> <INSDFeature_ key>source</INSDFeature key> <INSDFeature lecation>1. 42</INSD¥Feature location> —<INSDiFeature guals> —<INSDaalifier> <ENSDGualifier name>mol type</INSDualifier name> <ENSDQualifier value>other RNA<INSDQualifier value> <ENSDGualifier> —<INSDualifier id="q23"> <INSBOnslifier name>note</INSDQualifier name> <INSDBQualifier value>Sequence of crRNA3</ENSDM Jualifier value> <INSDQualifier> —<INSD (Qualifier id="q2"> <ENSDQualifier name>organism</INSD Qualifier name> <INSBQualifier value>synthetic construct</INSB(}ualifier value> </ENSDQualifier> </INSD Feature gquals> </INSDFeature> </INSDSeg feature-table> <INSBseg sequence>taatttctactaagtgtagatttgeggtagggetootteottc</ENSIMSeq sequence>

SJENSDSeg>

SSequenceara> —<SeguenceData sequenceIDNumber="2"> —<INSDSeg> <INSDSeg lengih>31JINSDseg length> <IANSDSeg moliype>DNA</INSD Seq maliype>

<INSBSeq divisien>PATZ/INSBDSeg divisien> —<ENSBSeg feature-table> —<{NSDFeature> <INSD¥Feature key>source</INSD¥Feature key> <INSDFeature location>]. 31</INSD Feature location> —<INSD Feature _guals> —<INSD{ualifier> <ENSDQualifier name>mol tpeJINSBi}salifier name> <INSBQualifier value>other DNA</INSDpualifier value> </ENSDQualifier> —<INSDPQualifier id="q24"> <ENSDQualifier name>note</INSB Qualifier name> <INSDQualifier value>RAA forward primer</INSD Qualifier value> </ENSDOualifier> —<INSD Qualifier id="g4"> <INSBQualifier name>organism</INSDQualifier name> <ENSP Qualifier value>synthetic construct</INSB Qualifier value> <INSDualifier> </INSD¥Featurs guals> </ENSD¥Feature> </INSD Seq feature-table> <INSBSeq sequence>gcctgctaattagtgcgttegtccactgegcINSBSeg sequence>

SENS Diseg> </SegquenceData> —<Seguence Data sequenceIDNumber="3"> —<INSDSeg> <INSDSeg length>31</INSDSeqg length> <INSDseg melfype>DNAJINSDSeg muoltype> <INSDSeg divisien>PATZENSDSeg divisien> —<INSDSeg feature-table> —<INSDFeature> <INSDFeature key>source</INSDFeature key> <INSDFsature location>]1. 31</INSDFeature location> —<iN5D Feature guals> —<INEBQualifier> <ENSDQualifier name>mol type</INSDGualifier name> <INSBPualifier valpe>other DNA</INSD Qualifier valwe> </ENSDOualifier> —<INSD Qualifier id="g25"> <INSBQualifier name>note</INSDBGualifier name> <ENSPBQualifier value>RAA reverse primer</INSD Qualifier value> <INSDualifier> —<INSDB{}ualifier id="g&"> <INSPualifier nawme>organism</INSD Qualifier nawe> <ENSDGualifier value>synthetic construct</INSB Qualifier vaine> </[ENSDOualifier> </INSD Feature guals> </EINSDFeature> </INSDSeq feature-table> <INSDSeq seguence>ctgttattgetctatctcgegeggcettetece</ INS eq sequencs> </INSDSeg> </Sequencelrata> —<SeguenceData sequenceIDNumber="4"> —<INSDSeg> <INSDseg length>21</INSDSeqg length>

<INSDSeg melfype>DNAJINSDSeg moltype> <INSDSeg divisien>PATSENSBDSeg divisien> —<ijSDseg festure-tabie> —<INSDFeatuvre> <INSDFeature key>source</INSDFeature key> <INSDFeature location>]. 21</INSDFeatave location> —<iNSDFeature guals> —<INSDQualifier> <ENSD Qualifier name>mol type</INSD Qualifier name> <ENSDPQualifier value>other DNA</ENSD Qualifier value> <INSDualifier> —<INSD Qualifier id="g26"> <INSPualifier name>note</INSD Qualifier name> <ENSDGualifier value>Upstream sequence of universal primer</INSDualifier valne> </ENSBGualifier> —<INSD{ualifier id="q8"> <INSDOnslifier name>organism</INSDGualifier name> <INSBQualifier value>synthetic construct</INSB{jualifier value> <INSDQualifier> </INSD¥eature guals> </INSD Features> </INSDSeq featuvre-table> <INSDseg sequence>tgttggtogoccggtcagtat</INS Seq sequsnes> </ENSDSeq> </Sequencellata> —<Seguencelata sequence] DNumber=":5"> —<INSDSeg> <INSDSeg lengih>22</INSDSeqg length> <INSBSeg moltype>DNA</INSDSeqg moliype> <INSDseg division>PATZENSBSeqg division> —NSDseg Testure-{abie> —<INSDFernire> <INSDFeature_ key>source</INSDFeature key> <INSDFeature lecation>1. 22</INSD¥Feature location> —<INSDFeature guals> —<INSDaalifier> <ENSDGualifier name>mol type</INSDualifier name> <ENSDQualifier value>other DNA</INS DB pualifier value> </ENSBGualifier> —<INSDualifier id="q27"> <INSBOnslifier name>note</INSDQualifier name> <INSDQualifier value>Downstream sequence of universal primer</INSDQualifier value> </ENSDOualifier> —<INSD Qualifier id="g10"> <ENSDQualifier name>organism</INSD Qualifier name> <ENSP Qualifier value>synthetic construct</INSB Qualifier value> </[ENSDOualifier> </INSD¥Featurs guals> </INSDFeature>

SINSDSeg feature-table> <INSDSeg sequsnee>cggcttetcccgcttgtccctc /INSBSeg seguence™>

SENS Diseg> </SegquenceData>

—<SeguenceData sequenceIDNumber="5"> —<INSDSeg> <INSDSeg length>42</INSDSeq length> <INSDseg moltype>RNA</INSDSeq meltype> <INSDSeg division>PAT</INSDSeqg divisien> —<INSDSeg feature-table> —<INSDFeature> <INSDFeature key>source</INSDFeature key> <INSDFeature location>]. 42</INSDFeatave location> —<iN5D Feature guals> —<INEBQualifier> <ENSDQualifier name>mol type</INSDGualifier name> <INSP Qualifier valpe>other RNA</INS DR ualifier value> </ENSDOualifier> —<INSD Qualifier id="g28"> <INSBQualifier name>note</INSDBGualifier name> <ENSDB Qualifier value>Sequence of crRNA1</INSD dualifier value> <INSDualifier> —<INSD Qualifier id="g12"™> <INSPualifier nawme>organism</INSD Qualifier nawe> <ENSDGualifier value>synthetic construct</INSB Qualifier vaine> </[ENSDOualifier> </INSD Feature guals> </ENSDF¥eature> </INSDSeq feature-table> <INSDSeqg seguence>taatttctactaagtgtagatcgcetcttgeectetectetce</INS Seq sequence> </INSDSeg> </Sequencelrata> —<SeguenceData sequenceIDNumber="7"> —<INSDSeg> <INSDseg length>42</INSDSeq length> <INSDSeg meltype>RNAINSDSeqg moltype> <INSDSeg division>PAT</ENSDSeyg division> —<ijSDseg feature-table> —<INSDFeatuvre> <INSDFeature key>source</INSDFeature key> <INSDFeature Jocatiom> 1 42</INSDFeatave location> —<ENSBEeature guals> —<INSDualifler> <ENSD Qualifier name>mol type</INSD Qualifier name> <ENSDPQualifier value>other RNA</INS BG ualifier value> <INSDualifier> —<INSD Qualifier id="g29"> <INSPualifier name>note</INSD Qualifier name> <ENSDualifier value>Sequence of crRNA2</ENST (Qualifier value> </[ENSDOualifier> —<INSDOgalifier id="g14"™> <ENSPQualifier name>organism</INSDQualifier name> <ENSDQualifier value>synthetic construct</INSB Qualifier value> <[ENSDBOualifier> </IMNSDFeature goals> </INSDFeature> </INSDSeq feature-table> <INSBSeg sequence>taatttctactaagtgtagatctegetegttecgetetettge</INSDS ey seqnenre> </EINSDSeg>

</SeqguenceData> —<Seguenceliata sequenceIDNumber="3"> —NSDSeg> <INSDseg lengih>42</INSDSeq length> <INSDSeg molype>RNAJINSDSeg maliype> <INSDSeg division>PAT</INSBSeq division> —<iNSiiseq feature-fabie> —<INSDFeatuve> <INSDFeature key>source/INSBFeatare key> <INSDFeature location 1 42</INSDFeature location> —INSDFestare gusls> —<INSDualifler> <INSDQualifier name>mol type</INSDQualifier name> <ENSDGualifier value>other RNA</ENSDGualifier value> </[ENSDOualifier> —<INSDualifier id="¢30"> <ENSPQualifier name>note</INSDQualifier name> <INSDOnslifier_ value>Seqence of crRNA4</INSD Qualifier value> <[ENSDBOualifier> —<INSIH}ualifier id="g18"> <ENSDGualifier name>organism</INSDGualifier name> <ENSDQualifier value>synthetic construct</INSB Qualifier value> <ENSDGualifier> </INSD¥eature guals> </INSDFeature> </INSDSeq feature-table> <INSDSeg seguence>taatttctactaagtgtagatgegttggccegegggtgcgge</INSR Seg sequence> </ENSDSeg> </SequenceData> —<SeguenceData sequenceIDNumber="9"> —<INSDSeg> <INSDSeg lengih>26J/INSDSeg length> <INSBSeg moltype>DNA</INSD Seg moltype> <INSDSeg division>PAT</INSBSeqg division —<INSDiseg Testore-fable> —<INSDFeature> <INSDFeature key>source</INSDFeatave key> <INSD Feature lecation™>]1. 26</INSD Feature location> —<INSDFeature guals> —<INSDualifier> <ENSPQualifier name>mol type</INSD Qualifier name> <INSB(ualifier value>other DNA</INSD{}ualifier value> <[ENSDBOualifier> —<INSIH}ualifier id="g3 "> <ENSDGualifier name>note</INSDQualifier name> <ENSDQualifier value>Primer cox 1-F</INSBualifier value> <ENSDGualifier> —<INSDualifier id="q20"> <ENSDQualifier name>organism</INSD Qualifier name> <INSBQualifier value>synthetic construct</INSB{jualifier value> <INSDQualifier> </INSD Feature quals> </INSD Features> </INSDSeq featuvre-table> <INSDseg sequence>agttactgctaataattttgtgtcat</INS Seq sequence>

</ENSDSeq> </Sequencellata> —<Seguencelata sequencelDNumber="10"> —<INSDSeg> <INSDSeg lengih>23</INSDSeqg length> <INSDSeg moliype>DNA</INSD Seq moliype> <INSDseg division>PATZENSBSeqg division> —NSDseg Testure-{abie> —<INSDFesture> <INSDFeature_ key>source</INSDFeature key> <INSDFeature lecation>1. 23</INSD¥Feature location> —<INSDFeature guals> —<INSDaalifier> <ENSDGualifier name>mol type</INSDualifier name> <INSPQualifier vahse>other DNA</INS DB ualifier value> <ENSDGualifier> —<INSDualifier id="q32"> <INSBOnslifier name>note</INSDQualifier name> <INSDQualifier value>Primer cox 1-R</INST(ualifier value> <INSDQualifier> —<INSD Qualifier id="q22"> <ENSDQualifier name>organism</INSD Qualifier name> <INSBQualifier value>synthetic construct</INSB(}ualifier value> </ENSDOualifier> </INSD Feature gquals> </INSDFeature> </INSDSey featuretable> <INSBseg sequence>atgatgtaaaaggcaaataaacc</INSB Seq sequence>

SJENSDSeg>

SSequenceara> </STi6Sequencelisting™>

Claims

CONCLUSIONS

1. Clustered regularly interspaced short palindromic repeat ribonucleic acid (crRNA) for echinococcal testing based on recombinase-assisted amplification (RAA) combined with CRISPR-Casl2a, wherein a nucleotide sequence is shown as SEQ ID NO:1.

A kit for testing echinococci based on RAA combined with CRISPR-Casl2a, comprising the crRNA of claim 1, cCasl2a4, a single-stranded DNA fluorophore quencher (ssDNA-FQ) and RAA primers.

The kit of claim 2, wherein the RAA primers comprise a forward primer having a nucleotide sequence shown as SEQ ID NO:2 and a reverse primer having a nucleotide sequence shown as SEQ ID NO:3.

The kit of claim 2, wherein a nucleotide sequence of the ssDNA-FQ is represented as a fluorophore TTATT quencher group.

The kit of any one of claims 2 to 4, further comprising positive plasmids, wherein the positive plasmids comprise a recombinant plasmid containing an 18s rRNA sequence from Echinococcus multilocularis and a recombinant plasmid containing an 18s rRNA sequence from Echinococcus granulosus.

The kit of claim 5, wherein amplification primers for the 18s rRNA sequence of Echinococcus multilocularis or the 18s rRNA sequence of Echinococcus granulosus comprise a forward primer having a nucleotide sequence shown as SEQ ID NO:4 and a reverse primer having a nucleotide sequence shown as SEQ ID NO:5.

Use of the crRNA of claim 1 combined with Casl2a, an ssDNA-FQ and RAA primers in the preparation of a kit for testing echinococci, wherein the RAA primers comprise a forward primer having a nucleotide sequence shown as SEQ ID NO:2 and a reverse primer having a nucleotide sequence shown as SEQ ID NO:3, and the echinococci comprise Echinococcus multilocularis and/or Echinococcus granulosus.

A method for testing Echinococcus multilocularis and/or Echinococcus granulosus for non-disease diagnostic purposes, comprising the steps of: employing an RAA technology to amplify the 18S5 rRNA sequence of a sample to be tested to obtain amplification products; and mixing the amplification products with the crRNA of claim 1, Casl2a and an ssDNA FQ to perform a CRISPR-Casl2Za reaction, assaying a fluorescent signal, and determining whether infection with Echinococcus multilocularis and/or Echinococcus granulosus occurs in the tested sample based on the presence or absence of the fluorescent signal.

The method of claim 8, wherein a reaction is carried out at 39°C for 15-25 minutes during amplification using RAA technology.

The method of claim 8, wherein during the CRISPR-Casl2a reaction, a working concentration of the crRNA is 100-800 µM, a working concentration of Casl2a is 400-800 µM, and a working concentration of the ssDNA-FQ is 750-850 µM; and the CRISPR-CaslZa reaction is carried out at 37°C for 15-25 minutes. -0-70-0-