CN113005230A - Asia Zika virus RT-RIA/CRISPR-Cas12a detection kit and detection method thereof - Google Patents

Asia Zika virus RT-RIA/CRISPR-Cas12a detection kit and detection method thereof Download PDF

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CN113005230A
CN113005230A CN202110466902.4A CN202110466902A CN113005230A CN 113005230 A CN113005230 A CN 113005230A CN 202110466902 A CN202110466902 A CN 202110466902A CN 113005230 A CN113005230 A CN 113005230A
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陈�峰
吴海磊
唐晓宇
杨国平
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Nantong Customs Comprehensive Technical Center Nantong Branch Of Jiangsu International Travel Health Care Center And Nantong Customs Port Outpatient Department
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Abstract

The invention relates to an Asia Zika virus RT-RIA/CRISPR-Cas12a detection kit and a detection method thereof. The kit comprises a primer and a probe sequence for Asian Zika virus RT-RIA/CRISPR-Cas12a detection, wherein the primer and the probe sequence comprise an RIA primer sequence of Zika virus ZIKV and a reference gene beta-actin gene, a Cas12a reaction crRNA sequence and a fluorescent report probe sequence, the 5 ' end of the crRNA sequence contains a T7 promoter recognition site, a fluorescent group marked at the 5 ' end of the ZIKV and beta-actin gene fluorescent report probe sequence is one of FAM, HEX, ROX, VIC and CY5, and a quenching group marked at the 3 ' end of the KV ZIKV and beta-actin gene fluorescent report probe sequence is one of TAMRA, MGB, BHQ1 and BHQ 2; and the 5' -end marked fluorescent groups of the probes ZIKV and beta-actin are different; the kit further comprises: a RIA reactive reagent component; cas12a reagent components; a sample-releasing agent; the RIA reaction reagent component comprises RIA Buffer, RIA enzyme and RIA activator; the Cas12a reagent component includes Cas Buffer and Cas12a enzyme.

Description

Asia Zika virus RT-RIA/CRISPR-Cas12a detection kit and detection method thereof
Technical Field
The invention relates to the technical field of detection, and particularly relates to an Asian Zika virus RT-RIA/CRISPR-Cas12a detection kit and a detection method thereof.
Background
Zika Virus (ZIKV) is an important mosquito-borne Virus belonging to the Flaviviridae family, the flavivirus genus, with single-stranded positive-strand RNA as the genome, a particle diameter of 40-50nm, and an Aedes mosquitos as the transmission medium. By 3 months in 2018, 84 ZIKV infection cases are reported in the world. 2016, 2 months, the first overseas input ZIKV infection case in mainland China is diagnosed[1]Subsequent sequential appearance of the input disease cases in Guangdong, Zhejiang, Beijing, etc[2]. The virus tends to spread in regions such as south-east Asia. The temperature and humidity in southern areas of China are very suitable for the growth and reproduction of mosquitoes, so that mosquito-borne infectious diseases are in a high-incidence state for a long time. The World Health Organization (WHO) has concluded that neonatal microcephaly, Guillain-Barre syndrome (Guillain-Barre syndrome) may be associated with Zika virus infection[3]. Serological diagnosis is difficult because Zika virus cross-reacts with other flaviviruses such as dengue virus, West Nile virus and yellow fever virus. The establishment of a rapid, simple, convenient and sensitive laboratory nucleic acid detection method has important significance for timely clinical diagnosis and treatment and epidemiological investigation.
The nucleic acid isothermal amplification technology has no need of repeated thermal denaturation and special instruments, has faster reaction speed, is suitable for on-site rapid detection, and has been widely applied to life science research and related fields[4]. More than 10 isothermal nucleic acid amplification technologies exist, wherein Recombinant Polymerase Amplification (RPA) is an isothermal nucleic acid amplification technology developed by TwistDx Inc., UK in 2006[5]Has the advantages of high sensitivity, strong specificity, rapid and convenient operation and the like, can realize quantitative analysis, has wide application prospect in many fields such as disease diagnosis, pathogen identification and the like, and is already used for detecting animal epidemic diseases such as foot-and-mouth disease virus[6]Peste des petits ruminants virus[7]Porcine parvovirus[8]Bovine viral diarrhea[9]Detection of viral hemorrhagic fever[10]Such as Marburg virus, Ebola virus, dengue virus, yellow fever virus and smallpox virus. Although the RPA amplification can be combined with the probe to carry out real-time fluorescence quantitative PCR detection, the method has extremely high requirements on primer and probe combination screening, and compared with the real-time fluorescence quantitative PCR method on the current market, the detection sensitivity is not substantially improved.
CRISPR is an abbreviation for regularly interspaced clustered short palindromic repeats (CRISPR), and Cas is an abbreviation for CRISPR associated protein (Cas). CRISPR/Cas was originally found in bacteria and is the defence system CRISPR-Cas detection system used by bacteria to recognize and destroy phage and other pathogen invasion. Gootenberg et al[11-13]A specific high-sensitivity enzymatic reporter assembly (SHERLOCK) based on cas13a and a sample inactivating nuclease technology (HUDSON) detection platform without nucleic acid extraction and heat treatment are established in sequence. Chen et al[14]Based on Cas12a, a DNA endonuclease targeted CRISPR trans-reporter (DETECTRR) detection system is established, and single-molecule-level sensitivity is realized. It is demonstrated that the CRISPR technique has gained some application in molecular diagnostic techniques.
Based on the requirement of national port on Zika virus prevention and control, the invention develops a set of Zika virus detection reagent based on RT-RIA/CRISPR-Cas12a fluorescence detection technology and a method thereof by taking simple, instant and high-sensitivity detection as a target, and provides an optional method for detecting and monitoring mosquito-borne infectious disease epidemic situation.
Disclosure of Invention
Aiming at the problems in the background technology, the invention provides an Asian Zika virus RT-RIA/CRISPR-Cas12a detection kit and a detection method thereof, which comprise a RT-RIA/CRISPR-Cas12 a-based fluorescence detection primer probe set, a kit and a detection method thereof.
ZIKV is called Zika virus for short;
RIA reaction and RT-RIA reaction are both the same system;
cas, Cas12a and CRISPR-Cas12a reaction show that the reaction systems are the same system;
the crRNA and the CrRNA are the same substance;
in order to achieve the above purpose, the invention adopts the technical scheme that: an Asia Zika virus RT-RIA/CRISPR-Cas12a detection kit comprises a primer and a probe sequence for Asia Zika virus RT-RIA/CRISPR-Cas12a detection, wherein the primer and the probe sequence comprise an RIA primer sequence of Zika virus ZIKV and an internal reference gene beta-actin gene, a Cas12a reaction crRNA sequence and a fluorescence report probe sequence,
the nucleotide sequence of the ZIKV RIA primers is shown below:
and (3) primer ZIKV-F:
5’-TTGAGGGAGAGTTCAAGCTTAGGACGGAGCAA-3’，(SEQ ID NO：1)；
and (3) primer ZIKV-R:
5’-GAGATGCGACCTGATAGGCCAGCCAAACAGGA-3’，(SEQ ID NO：2)；
primer beta-actin-F:
5’-TGGATCAGCAAGCAGGAGTATGACGAGTCCGG-3’，(SEQ ID NO：3)；
primer beta-actin-R:
5’-CATCTTGTTTTCTGCGCAAGTTAGGTTTTGTC-3’，(SEQ ID NO：4)；
the 5' end of the crRNA sequence contains a T7 promoter recognition site, and the nucleotide sequence is shown as follows:
ZIKV-crRNA：
5’-TAATTTCTACTAAGTGTAGATgacctttgtggaactcatgaaaagaggagatctt-3’，(SEQ ID NO：5)；
β-actin-crRNA：
5’-TAATTTCTACTAAGTGTAGATcggtggacgatggaggggccgg-3’，(SEQ ID NO：6)；
the nucleotide sequence of the fluorescent reporter probe is shown as follows:
ZIKV-P：
5’-TTATT-3’，(SEQ ID NO：7)；
β-actin-P：
5’-TTAATT-3’，(SEQ ID NO：8)；
the fluorescent group marked at the 5 'end of the ZIKV and beta-actin gene fluorescent reporter probe sequence is one of FAM, HEX, ROX, VIC and CY5, and the quenching group marked at the 3' end of the ZIKV and beta-actin gene fluorescent reporter probe sequence is one of TAMRA, MGB, BHQ1 and BHQ 2; and the 5' -end marked fluorescent groups of the probes ZIKV and beta-actin are different;
the kit further comprises:
(1) a RIA reactive reagent component;
(2) cas12a reagent components;
(3) a sample-releasing agent;
the RIA reaction reagent component comprises RIA Buffer, RIA enzyme and RIA activator;
the Cas12a reagent component includes Cas Buffer and Cas12a enzyme.
Furthermore, FAM and VIC fluorescein are respectively marked at the 5 'ends of the ZIKV and beta-actin gene fluorescent reporter probe sequences, and BHQ1 quenching groups are respectively marked at the 3' ends of the ZIKV and beta-actin gene fluorescent reporter probe sequences.
Furthermore, in an RIA reaction system, the final concentration of the ZIKV and beta-actin gene primers is 400 nmol/L; the concentration of crRNA in the Cas12a reaction system is 70nmol/L, and the concentration of the fluorescent reporter probe is 500 nmol/L.
Further, the RIA reaction and the Cas12a reaction are carried out under the same reaction conditions and are carried out at 42 ℃, wherein the RIA reaction time is 15min, and the Cas12a reaction time is 10 min.
Further, the RIA activator is MgOAc; the RIA enzyme comprises reverse transcriptase, RNase inhibitor, recombinase combined with single-stranded nucleic acid, single-stranded DNA binding protein and strand displacement DNA polymerase; the Cas12a enzyme is LbCas12 a.
Further, the final concentration of the RIA activator MgOAc in the RIA reaction system is 14 mmol/L; the concentration of LbCas12a enzyme in the Cas12a reaction system was 1. mu.M.
Further, the RIA Buffer comprises a nucleic acid releasing agent, a metal chelating agent, a buffering agent, a surfactant, a nucleic acid stabilizing agent, a cell membrane penetrating agent and a nuclease inhibitor; the nucleic acid releasing agent is sodium lauroyl sarcosinate, the buffer solution is Tris-HCl, the metal chelating agent is EDTA, the surfactant is Triton X-100, the cell membrane penetrating agent is DMSO, the nucleic acid stabilizing agent is DTT, and the nuclease inhibitor is DEPC water.
Still further, the RIA Buffer comprises sodium lauroyl sarcosinate with the concentration of 70 mM-170 mM, Tris-HCl with the concentration of 50mM, EDTA with the concentration of 20 mM-50 mM, Triton X-100 with the concentration of 30 mM-75 mM, DMSO with the concentration of 1.2M-3.6M, and DTT with the concentration of 5 mM-10 mM.
An Asian Zika virus RT-RIA/CRISPR-Cas12a detection method, comprising the following steps:
(1) preparing an RIA reaction system and a Cas12a reaction system;
(2) RIA reaction;
(3) cas12a reaction;
(4) and (6) judging the result.
Further, an Asia Zika virus RT-RIA/CRISPR-Cas12a detection method comprises the following steps:
(1) preparation of reagents
Preparing a RIA reaction system: preparing 50 mu L of RIA reaction system, wherein 30.0 mu L of RIA Buffer, 10.0 mu L of RIA enzyme and 2.0 mu L of each of upstream and downstream RIA of 10 mu mol/L in the RIA reaction system are added with DEPC water to be supplemented to 45.5 mu L, the mixture is evenly mixed and then placed on ice, 2.0 mu L of serum template is added, and 2.5 mu L of MgOAc RIA activator with the concentration of 280mmol/L is added;
cas12a reaction system was configured: 3 mu L of Cas12a Buffer, 3 mu L of 300nM crRNA, 1 mu M LbCas12a 1 mu L, 1.5 mu L of 10 mu M FQ fluorescent reporter probe, supplementing water to 27 mu L and taking 3 mu L of RIA reaction product as a template;
(2) fluorescence detection
The RIA reaction is carried out on constant temperature equipment or variable temperature PCR equipment, and the reaction conditions are as follows: 15min at 37 ℃; cas12a fluorescence detection is carried out on constant-temperature or variable-temperature real-time fluorescence detection equipment containing FAM or VIC channels, and the reaction conditions are as follows: fluorescence was collected at 37 ℃ for 10min every 0.5 min.
(3) Analysis of results
Quality control standard: the VIC channel is positive, and the fluorescence curve is an obvious S-shaped curve, which indicates that the nucleic acid extraction and amplification reaction is normal;
and (4) interpretation of results: analyzing the result on the premise of qualified quality control, and if an FAM channel has an obvious S-shaped curve or an amplification curve has an exponential growth trend, determining that the result is positive for the Zika virus; otherwise, the result is negative.
By adopting the scheme, the invention has the beneficial effects that:
(1) sample direct amplification: because the RT-RIA reaction Buffer contains the reagent components capable of directly extracting nucleic acid, the serum sample does not need to be extracted by virus RNA, and the separated serum can be directly added into an RT-RIA reaction system for RT-RIA amplification;
(2) the requirement on the instrument is simple: RT-RIA detection and CRISPR-Cas12a detection only need constant temperature fluorescence detection equipment, do not need variable temperature PCR equipment, and are suitable for miniaturized constant temperature fluorescence detectors of various models.
(3) The sensitivity is high: the lowest detection sensitivity is 5.0 multiplied by 102copies/mL, namely each PCR reaction can detect Zika virus RNA with the concentration as low as 1.5copies, and single copy detection of the Zika virus is realized;
(4) the specificity is good: specifically detecting Zika virus nucleic acid, and has no detection signal for other virus nucleic acids such as West Nile virus, dengue virus (I-IV), yellow fever virus, hepatitis C virus, etc.
(5) The repeatability is good: the test results of 10 times of repeated tests on the pseudovirus standard products with the concentrations of 1.0X 107copies/mL and 1.0X 104copies/mL are positive, and the variation coefficient of log (fluorescence intensity response value) is less than 5 percent.
Drawings
FIG. 1-1 is a diagram showing the results of screening different combinations of ZIKV RIA primers, and FIG. 1-2 is a diagram showing the results of screening combinations of β -actin RIA primers;
FIG. 2-1 is a graph showing the influence of different RIA primer concentrations on the detection sensitivity of ZIKV gene, and FIG. 2-2 is a graph showing the influence of the detection sensitivity of β -actin gene;
FIG. 3 is a graph of the effect of different gRNA concentrations on ZIKV detection sensitivity;
FIG. 4 is a graph of the effect of different LbCas12a enzyme concentrations on ZIKV detection sensitivity;
FIG. 5 is a graph of the effect of different FQ fluorescent probe concentrations on ZIKV detection sensitivity;
FIG. 6 is a graph showing the effect of measurements made at different reaction times;
FIG. 7 is a graph showing the results of the ZIKV sensitivity assay;
FIG. 8 is a diagram showing the results of specific detection by the kit;
FIG. 9 is a graph showing the results of a serum simulation test.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
The invention discloses a (Asia-type) Zika virus RT-RIA/CRISPR-Cas12a fluorescent detection primer probe set, a kit and a detection method.
Wherein, the primer probe sequence is shown as SEQ ID NO: 1 to SEQ ID NO: shown in fig. 8.
The invention discloses a (Asia type) Zika virus RT-RIA/CRISPR-Cas12a fluorescence detection kit, which mainly comprises: (Zika virus) RT-RIA reaction reagent and (Zika virus) RT-RIA primer, CRISPR-Cas12a reaction reagent and Cas primer probe.
The kit can realize the rapid constant-temperature detection of the Zika virus, and the detection system has the lowest detection limit of 5.0 multiplied by 102copies/mL to the Zika virus, which is higher than the RT-qPCR detection sensitivity; specifically detecting Zika virus nucleic acid, and has no detection signal for other virus nucleic acids such as West Nile virus, dengue virus (I-IV), yellow fever virus, hepatitis C virus and the like; the test results of 10 times of repeated tests on the pseudovirus standard products with the concentrations of 1.0 × 107copies/mL and 1.0 × 104copies/mL are positive, and the variation coefficient of log (fluorescence intensity response value) is less than 5 percent; the simulation test result shows that the detection results are positive when the concentration of the ZIKV pseudovirus exceeds 5.0 multiplied by 102 copies/mL; the comparison test result shows that the detection coincidence rate of the ZIKV RT-RIA/CRISPR-Cas12a detection method on clinical samples is 100%.
Namely, the primer (and) probe sequence (group) provided by the invention comprises an RT-RIA primer sequence (sequence) group detected by ZIKV RT-RIA/CRSPR-Cas12a and a primer probe (sequence) group detected by CRISPR-CAS12a, wherein the nucleotide sequence of the ZIKV RT-RIA primer is shown as follows:
and (3) primer ZIKV-F:
5’-TTGAGGGAGAGTTCAAGCTTAGGACGGAGCAA-3’，(SEQ ID NO：1)；
and (3) primer ZIKV-R:
5’-GAGATGCGACCTGATAGGCCAGCCAAACAGGA-3’，(SEQ ID NO：2)；
primer beta-actin-F:
5’-TGGATCAGCAAGCAGGAGTATGACGAGTCCGG-3’，(SEQ ID NO：3)；
primer beta-actin-R:
5’-CATCTTGTTTTCTGCGCAAGTTAGGTTTTGTC-3’，(SEQ ID NO：4)；
the CRRNA sequence 5' end in the CRISPR-CAS12a reaction system contains a T7 promoter recognition site, and the nucleotide sequence is shown as follows:
ZIKV-crRNA：
5’-TAATTTCTACTAAGTGTAGATgacctttgtggaactcatgaaaagaggagatctt-3’，(SEQ ID NO：5)；
β-actin-crRNA：
5’-TAATTTCTACTAAGTGTAGATcggtggacgatggaggggccgg-3’，(SEQ ID NO：6)；
the CrRNA primer is a part of a target fragment for RT-RIA amplification, and the 5' end of the CrRNA primer contains a T7 promoter sequence which can be used as a T7 RNA polymerase recognition site in CRISPR-CAS12a reaction.
The nucleotide sequence of the fluorescent reporter probe is shown as follows:
ZIKV-P：
5’-TTATT-3’，(SEQ ID NO：7)；
β-actin-P：
5’-TTAATT-3’，(SEQ ID NO：8)；
the fluorescent group marked at the 5 'end of the probe sequence is one of FAM, HEX, ROX, VIC and CY5, and the quenching group marked at the 3' end of the probe sequence is one of TAMRA, MGB, BHQ1 and BHQ 2; preferably, 5 '-end labeled fluorophores of ZIKV and beta-actin are FAM and VIC fluorescein respectively, and 3' -end of the probe is labeled with BHQ1 quenching group.
Based on the primer probe group, the invention provides an Asia Zika virus RT-RIA/CRISPR-Cas12a kit which has high sensitivity, strong specificity and good repeatability, the kit contains the primer probe group, and the final concentration of the RIA primer probe in an RT-RIA system is 400 nmol/Lol/L; the concentrations of CrRNA and (fluorescent) reporter probe in the CRISPR-Cas12a reaction system were 70nmol/L and 500nmol/L, respectively.
Further, the kit should also contain an RT-RIA reaction reagent component and a CRISPR-Cas12a reaction reagent component. The RT-RIA reaction reagent (component) comprises RIA Buffer, RIA enzyme and RIA activator; CRISPR-Cas12a reagents (components) include Cas Buffer and Cas12a enzymes.
The RIA Buffer comprises a nucleic acid releasing agent, a metal chelating agent, a buffering agent, a surfactant, a nucleic acid stabilizer, a cell membrane penetrating agent and a nuclease inhibitor.
Further, the nucleic acid releasing agent, the metal chelating agent, the buffering agent, the surfactant, the nucleic acid stabilizer, the cell membrane permeation agent and the nuclease inhibitor are sodium lauroyl sarcosinate, EDTA, Tris-HCl, Triton X-100, DTT, DMSO and DEPC water respectively, and the final concentration of the sodium lauroyl sarcosinate is 2-5 percent, the final concentration of the Tris-HCl is 50mM, the final concentration of the EDTA is 20-50 mM, the final concentration of the Triton X-100 is 2-5 percent, the final concentration of the DMSO is 10-30 percent, and the final concentration of the DTT is 5-10 mM. The conversion is uniform, namely the concentration of the sodium lauroyl sarcosinate is 70 mM-170 mM, the concentration of Tris-HCl is 50mM, the concentration of EDTA is 20 mM-50 mM, the concentration of Triton X-100 is 30 mM-75 mM, the concentration of DMSO is 1.2M-3.6M, and the concentration of DTT is 5 mM-10 mM.
The final concentration of RIA activator MgOAc in the RIA reaction system is 14 mmol/L.
The concentration of LbCas12a enzyme in the CRISPR-CAS12a reaction system is 1 μ M.
Based on the kit, the invention also provides a detection method of the kit, which comprises the following steps:
(1) preparing an RT-RIA reaction system and a CRISPR-Cas12a reaction system;
(2) RT-RIA reaction;
(3) CRISPR-Cas12a reaction;
(4) and (6) judging the result.
Preferably, the RT-RIA reaction system is: 50 mu L (RIA) reaction system RIA Buffer 30.0 mu L, RIA enzyme 10.0 mu L, 10 mu mol/L upstream and downstream RIA each 2.0 mu L, adding DEPC water to make up to 45.5 mu L, mixing, placing on ice, adding serum template 2.0 mu L, adding 280mmol/L MgOAc RIA activator 2.5 mu L.
Cas12a reaction system: cas12a Buffer 3. mu.L, 300nM crRNA 3. mu.L, 1. mu.M LbCas12a 1. mu.L, 10. mu.M FQ fluorescent reporter probe 1.5. mu.L, water supplemented to 27. mu.L, 3. mu.L of RPA reaction product as template.
Preferably, the RT-RIA reaction conditions are: 15min at 37 ℃; cas12a fluorescence detection can be carried out on constant-temperature or variable-temperature real-time fluorescence detection equipment containing FAM or VIC channels, and the reaction conditions are as follows: fluorescence was collected at 37 ℃ for 10min every 0.5 min.
Example 1: design and screening of RT-RIA primers
According to the bioinformatics analysis result and literature reports, Asian and African Zika virus genome sequences and dengue virus I-IV, yellow fever virus, West Nile virus, Japanese encephalitis virus and hepatitis C virus genome sequences are downloaded from NCBI databases, MEGA6.06 biological software is used for comparison, a conserved region of Asian Zika virus non-coding structural protein gene NS5 is selected as a target gene design primer, primer premier5.0 software is used according to the Twist primer design principle: 3-5 nucleotides at the 5' end of the primer should avoid poly-guanine; 3 bases at the 3' end are guanine and cytosine, which is beneficial to the stable combination of polymerase and can improve the amplification performance of the primer; it is preferred that no specific sequences, such as long stretches of polypurine or polypyrimidine, are present in the primer; too high (> 70%) or too low (< 30%) GC content is detrimental to RPA amplification; in addition, the primer should be designed to avoid sequences that are prone to form secondary structures, primer-primer interactions, and hairpin structures, and to reduce primer dimer formation. Respectively designing 4 groups of RPA amplification primers, performing Blast on the 4 groups of designed primers in NCBI, and comparing results to obtain Asia Zika viruses.
CRISPR gRNA sequences were designed on-line based on https:// ports. branched. organ. org/gpp/public/analysis-tools/sgrn-design, with primer sequences and markers as shown in Table 1 below.
TABLE 1 primer and Probe details
Table 1 Primer details
Figure BDA0003043534750000061
Figure BDA0003043534750000071
RT-RIA amplification reagent is adopted to screen 4 pairs of ZIKV and 4 pairs of beta-actin gene RPA primers, the reaction system and the reaction program refer to the research result of the earlier stage of the laboratory, 30.0 mu L of RIA Buffer, 10.0 mu L of RIA enzyme and 2.0 mu L of 10 mu mol/L upstream and downstream RIA in 50 mu L reaction system are respectively added with DEPC water to be supplemented to 45.5 mu L, the mixture is evenly mixed and then is moved on ice, 2.0 mu L of serum template is added, and 2.5 mu L of MgOAc RIA activator with the concentration of 280mmol/L is added. ZIKV pseudovirus standard (1.0 × 10) diluted by 10-fold gradient7copies/mL、1.0×106copies/mL、1.0×105copies/mL and 1.0X 104copies/mL) and 10-fold gradient diluted (10 ng/mu L, 1 ng/mu L, 100 pg/mu L and 10 pg/mu L) human serum RNA as templates, the reaction temperature is set at 37 ℃, the reaction time is 15min, the amplification product is detected by 1.5% agarose gel electrophoresis, and the optimal RPA primer combinations of the ZIKV gene and the beta-actin gene are respectively screened.
Example 2: optimization of reaction conditions
(1) RT-RIA primer concentration optimization
Setting 3 different primer concentration levels (the concentrations are respectively 200nmol/L, 400nmol/L and 600nmol/L), and diluting the ZIKV pseudovirus standard substance (1.0 multiplied by 10) by using a 10-fold gradient7copies/mL、1.0×106copies/mL、1.0×105copies/mL and 1.0X 104copies/mL) and 10-fold gradient dilutions (10 ng/. mu.L, 1 ng/. mu.L, 100 pg/. mu.L and 10 pg/. mu.L) of human serum RNA and negativeAnd (3) carrying out RT-RIA reaction by taking the control as a sample to be detected, detecting the amplification product by adopting 1.5% agarose gel electrophoresis, and testing the influence of different primer concentrations on the amplification result.
As a result: different concentrations of the RPA primer had an effect on the amplification sensitivity of ZIKV and beta-actin. When the final concentration of the RPA primer is 200nol/L, the detection sensitivity of the ZIKV and beta-actin genes is low, wherein the detection sensitivity of the ZIKV pseudovirus is 105The detection sensitivity of the gene of beta-actin is 100 pg/mu L; when the final concentration of the RPA primer is increased to 400nol/L, the detection sensitivity of the ZIKV gene and the beta-actin gene is respectively increased to 104copies/mL and 10 pg/. mu.L; when the concentration of the RPA primer is continuously increased to 600nol/L, the detection sensitivity is not increased (see the attached figures 2-1 and 2-2), and finally the final concentration of the RPA primer of the ZIKV and beta-actin genes in the RT-RIA system is determined to be 400 nol/L.
(2) gRNA concentration optimization
4 different gRNA concentration levels (concentrations of 30nmol/L, 50nmol/L, 70nmol/L and 100nmol/L, respectively) were set to add ZIKV pseudovirus to a final concentration of 1.0X 103Taking a copes/mL serum sample and a negative control as a sample to be tested to perform RT-RIA reaction, taking 3 mu L of RPA reaction product as a template, adding the product into a 27 mu LCas12a reaction system, processing 3 groups of parallel samples, and testing the influence of different gRNA concentrations on the detection result.
As a result: low concentrations of gRNA affect the detection sensitivity of ZIKV, with a final serum concentration of 1.0 × 10 at a gRNA concentration of 30nol/L3The detection result of the ZIKV pseudovirus of copies/mL is negative; when the concentration of gRNA is more than 50nol/L, the ZIKV detection result is positive (see figure 3). In consideration of the long-term storage stability, the final concentration of gRNA in the reaction system was finally determined to be 70 nol/L.
(3) LbCas12a concentration optimization
4 different LbCas12a concentration levels (concentration: 30nmol/L, 50nmol/L, 70nmol/L and 100nmol/L, respectively) were set to add ZIKV pseudovirus to a final concentration of 1.0X 103Taking a copes/mL serum sample and a negative control as a sample to be tested to perform RT-RIA reaction, taking 3 mu L of RPA reaction product as a template, adding the product into a 27 mu LCas12a reaction system, processing 3 groups of parallel samples, and testing different LbCas12a concentrationsInfluence on the detection result.
As a result: LbCas12a enzyme at 4 different concentrations for 1.0 × 103The detection results of the ZIKV pseudoviruses of copies/mL are positive. The LbCas12a enzyme with low concentration (30nmol/L) or high concentration (100nmol/L) can reduce the fluorescence intensity for detection, and the ZIKV and beta-actin genes have stronger fluorescence intensity and certain difference when the concentration of the LbCas12a enzyme is 50nmol/L or 70nmol/L, so that the detection is easy to distinguish (see figure 4). Considering the long-term storage stability, the concentration of LbCas12a enzyme in the reaction system is finally determined to be 70 nol/L.
(4) Concentration optimization of FQ fluorescent reporter probe
4 different FQ fluorescent reporter probe concentration levels (concentrations: 100nmol/L, 300nmol/L, 500nmol/L and 700nmol/L, respectively) were set to add ZIKV pseudoviruses to a final concentration of 1.0X 103Taking a copies/mL serum sample and a negative control as a sample to be tested to perform RT-RIA reaction, taking 3 mu L of RPA reaction product as a template, adding the RPA reaction product into a 27 mu LCas12a reaction system, processing 3 groups of parallel samples, and testing the influence of different LbCas12a concentrations on the detection result.
As a result: FQ fluorescent reporter probe pairs of different concentrations 1.0X 103The detection results of the ZIKV pseudoviruses of copies/mL are positive, but the fluorescence intensity of ZIKV detection increases with the increase of the concentration of the FQ fluorescent reporter probe. When the concentration of the FQ fluorescent reporter probe is 500nmol/L, the ZIKV and beta-actin genes have high fluorescent intensity and certain difference, and are easy to distinguish (see figure 5). The final concentration of the FQ fluorescent reporter probe in the reaction system is determined to be 500 nol/L.
(5) Cas12a reaction time test
The CRISPR-Cas12a reaction is distinguished from the PCR reaction by not relying on the Tm value of the primers, the reaction temperature is mainly dependent on the enzymatic kinetic reaction, and the reaction time is only required to be groped at the recommended optimal temperature of 37 ℃. According to the optimized reaction system, the concentration of the fixed gRNA is 70nmol/L, the LbCas12a 70 is 70nmol/L, the FQ fluorescent report probe is 500nmol/L, and the reaction temperature is 37 ℃. To add ZIKV pseudovirus to a final concentration of 5.0X 102Taking a copes/mL serum sample and a negative control as a sample to be detected to perform RT-RIA reaction, and adding 3 mu L of RPA reaction product as a template into a 27 mu LCas12a reaction systemAnd 3 parallels are arranged, fluorescence is collected in 5min, 10min and 15min respectively, and the minimum detection limit reference substance can be detected in the optimal reaction system within the required minimum time.
As a result: when the reaction time of the CRISPR-Cas12a system is within 5min, the concentration in serum is 5.0 multiplied by 102The detection result of the reference substance with the lowest detection limit of copies/mL is negative; when the reaction time is 10min, the detection result of the reference substance with the lowest detection limit is positive; the fluorescence intensity is basically consistent with that at 15min (see figure 6), and finally the optimal reaction time of the CRISPR-Cas12a is determined to be 10 min.
Example 3: evaluation of Performance
(1) Sensitivity test
Using 2.0 × 10 ZIKV pseudovirus standard3copies/mL、1.0×103copies/mL、5.0×102copies/mL) and 250X 102The copies/mL and the negative control are samples to be detected, each concentration sample and the negative control are repeated for 10 times, the constructed RPA/CRISPR-Cas12a method is adopted for detection, and the detection sensitivity is determined.
The experimental result shows that when the concentration of the ZIKV pseudovirus is 250copies/mL, the result is positive only 3 times after 10 times of repeated detection, and the positive rate is 30 percent (3/10); when the concentration of the ZIKV pseudoviruses is not lower than 500copies/mL, the detection results are positive, and the detection rate is 100 percent (10/10) (see figure 7). Therefore, the detection sensitivity of the RT-RIA/CRISPR-Cas12a fluorescence detection system constructed in the present study on ZIKV is 500copies/mL, i.e. 1.5 copies/reaction (3 μ LRT-RIA product as template).
(2) Specificity test
The specificity test is carried out by extracting nucleic acid from Zika virus nucleic acid, dengue virus (I-IV type) and yellow fever virus attenuated inactivated vaccine, hepatitis C virus serum, encephalitis B virus, parainfluenza virus, rhinovirus, respiratory syncytial virus A group, adenovirus 7 type, measles virus, rubella virus, mumps virus, influenza A virus H1N1, influenza B virus Yamagata, mycoplasma pneumoniae and chlamydia pneumoniae positive throat swab specimen and using the RT-RIA/CRISPR-Cas12a detection system constructed by the research.
The ZIKV nucleic acid amplification result is positive; the amplification results of the internal standard of the inactivated attenuated vaccines of the dengue viruses (I-IV) and the yellow fever viruses are negative; the amplification results of the internal standard of other pharyngeal swab samples are positive, the ZIKV detection results are negative, and no non-specific amplification (see figure 8) exists, which indicates that the reaction system has good specificity.
(3) Repeatability test
High concentration pseudovirus standard of ZIKV (1.0X 10)6copies/mL) and low concentration pseudovirus standard (1.0X 10)3copies/mL) and negative control were performed on the specimens in duplicate, 10 replicates per concentration. And (5) counting the variation coefficient of the log fluorescence intensity response value.
ZIKV virus pair high concentration (1.0X 10)6copies/mL) and low concentration (1.0X 10)3copies/mL) was positive, the coefficients of variation of the original fluorescence intensity were 4.77% and 2.48%, respectively, and the original fluorescence intensity was Log10The coefficient of variation of the (RF) values is 0.51% and 0.27%, and the coefficient of variation is within 5% (see Table 2 below), which shows that the RT-RIA/CRISPR-Cas12a fluorescence detection system constructed in the research has good repeatability.
TABLE 2 repeatability test results
Table 2 Results of reproducibility
Figure BDA0003043534750000101
(4) Serum analogue comparison test
ZIKV pseudoviruses are added into serum of healthy people to make the final concentration of the viruses be 1.0 multiplied by 10 respectively5copies/mL、1.0×104copies/mL、1.0×103copies/mL、5.0×102copies/mL、250×102And (3) processing the copies/mL and 1 part of control serum without addition for 3 times, respectively detecting the extracted nucleic acid by adopting the constructed ZIKV RPA/CRISPR-Cas12a detection method and the ZIKV RT-qPCR detection method, and comparing the detection sensitivity difference of the two.
The result of a serological simulation test shows that the detection sensitivity of RT-RIA/CRISPR-Cas12a to ZIKV in serum is high, when the concentration of pseudovirus added in serum is as low as 250copies/mL, the detection results of RT-qPCR are negative, and a positive signal can be detected by RT-RIA/CRISPR-Cas12 a; when the final concentration of the ZIKV pseudovirus is added to the serum to be more than 500copies/mL, positive results can be stably detected by the RT-RIA/CRISPR-Cas12a and the RT-qPCR method (see figure 9). The detection sensitivity of the ZIKV RT-RIA/CRISPR-Cas12a detection system constructed in the research on the ZIKV in the serum is 500 copies/mL.
(5) Clinical sample testing
100 pharyngeal swab samples which are detected in the past are detected by using a ZIKV RT-RIA/CRISPR-Cas12a detection system constructed in the research, the detection result and the detection result of a real-time fluorescence PCR detection kit in the past are subjected to statistical analysis, and the positive rate, the negative rate and the total coincidence rate of the two are compared.
After 100 cases of remaining throat swab specimens are detected in the past, the ZIKV RT-RIA/CRISPR-Cas12a detection system constructed by the method is used for detecting, only 1 case of Zika virus positive specimens are detected, and the positive coincidence rate, the negative coincidence rate and the total coincidence rate are 100%, which indicates that the detection system has reliable detection results on clinical specimens.
Figure BDA0003043534750000102
Figure BDA0003043534750000111
Figure BDA0003043534750000121
Figure BDA0003043534750000131
Figure BDA0003043534750000141
Figure BDA0003043534750000151
Sequence listing
<110> Nantong customs integrated technology center (Jiangsu international travel health care center-Nantong branch center, Nantong customs port outpatient department)
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aagtagcagc cattgaggga gagttcaagc ttaggacg 38
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taatttctac taagtgtaga tcggtggacg atggaggggc cgg 43

Claims (10)

1. An Asian Zika virus RT-RIA/CRISPR-Cas12a detection kit comprises a primer and a probe sequence for Asian Zika virus RT-RIA/CRISPR-Cas12a detection, wherein the primer and the probe sequence comprise an RIA primer sequence of Zika virus ZIKV and a reference gene beta-actin gene, a Cas12a reaction crRNA sequence and a fluorescence report probe sequence, and the detection kit is characterized in that:
the nucleotide sequence of the ZIKV RIA primers is shown below:
and (3) primer ZIKV-F:
5’-TTGAGGGAGAGTTCAAGCTTAGGACGGAGCAA-3’，(SEQ ID NO：1)；
and (3) primer ZIKV-R:
5’-GAGATGCGACCTGATAGGCCAGCCAAACAGGA-3’，(SEQ ID NO：2)；
primer beta-actin-F:
5’-TGGATCAGCAAGCAGGAGTATGACGAGTCCGG-3’，(SEQ ID NO：3)；
primer beta-actin-R:
5’-CATCTTGTTTTCTGCGCAAGTTAGGTTTTGTC-3’，(SEQ ID NO：4)；
the 5' end of the crRNA sequence contains a T7 promoter recognition site, and the nucleotide sequence is shown as follows:
ZIKV-crRNA：
5’-TAATTTCTACTAAGTGTAGATgacctttgtggaactcatgaaaagaggagatctt-3’，(SEQ ID NO：5)；
β-actin-crRNA：
5’-TAATTTCTACTAAGTGTAGATcggtggacgatggaggggccgg-3’，(SEQ ID NO：6)；
the nucleotide sequence of the fluorescent reporter probe is shown as follows:
ZIKV-P：
5’-TTATT-3’，(SEQ ID NO：7)；
β-actin-P：
5’-TTAATT-3’，(SEQ ID NO：8)；
the fluorescent group marked at the 5 'end of the ZIKV and beta-actin gene fluorescent reporter probe sequence is one of FAM, HEX, ROX, VIC and CY5, and the quenching group marked at the 3' end of the ZIKV and beta-actin gene fluorescent reporter probe sequence is one of TAMRA, MGB, BHQ1 and BHQ 2; and the 5' -end marked fluorescent groups of the probes ZIKV and beta-actin are different;
the kit further comprises:
(1) a RIA reactive reagent component;
(2) cas12a reagent components;
(3) a sample-releasing agent;
the RIA reaction reagent component comprises RIA Buffer, RIA enzyme and RIA activator;
the Cas12a reagent component includes Cas Buffer and Cas12a enzyme.
2. The Asian Zika virus RT-RIA/CRISPR-Cas12a detection kit according to claim 1, wherein: FAM and VIC fluorescein are respectively marked at the 5 'ends of the ZIKV and beta-actin gene fluorescent reporter probe sequences, and BHQ1 quenching groups are respectively marked at the 3' ends of the ZIKV and beta-actin gene fluorescent reporter probe sequences.
3. The Asian Zika virus RT-RIA/CRISPR-Cas12a detection kit according to claim 1, wherein in the RIA reaction system, the final concentration of ZIKV and beta-actin gene primers is 400 nmol/L; the concentration of crRNA in the Cas12a reaction system is 70nmol/L, and the concentration of the fluorescent reporter probe is 500 nmol/L.
4. The Asian Zika virus RT-RIA/CRISPR-Cas12a detection kit according to claim 1, wherein: the RIA reaction and the Cas12a reaction are carried out under the same reaction condition and are carried out at 42 ℃, wherein the RIA reaction time is 15min, and the Cas12a reaction time is 10 min.
5. The Asian Zika virus RT-RIA/CRISPR-Cas12a detection kit according to claim 1, wherein: the RIA activator is MgOAc; the RIA enzyme comprises reverse transcriptase, RNase inhibitor, recombinase combined with single-stranded nucleic acid, single-stranded DNA binding protein and strand displacement DNA polymerase; the Cas12a enzyme is LbCas12 a.
6. The Asian Zika virus RT-RIA/CRISPR-Cas12a detection kit according to claim 5, wherein: the final concentration of the RIA activator MgOAc in the RIA reaction system is 14 mmol/L; the concentration of LbCas12a enzyme in the Cas12a reaction system was 1. mu.M.
7. The Asian Zika virus RT-RIA/CRISPR-Cas12a detection kit according to claim 1, wherein: the RIA Buffer comprises a nucleic acid releasing agent, a metal chelating agent, a buffering agent, a surfactant, a nucleic acid stabilizer, a cell membrane penetrating agent and a nuclease inhibitor; the nucleic acid releasing agent is sodium lauroyl sarcosinate, the buffer solution is Tris-HCl, the metal chelating agent is EDTA, the surfactant is Triton X-100, the cell membrane penetrating agent is DMSO, the nucleic acid stabilizing agent is DTT, and the nuclease inhibitor is DEPC water.
8. The Asian Zika virus RT-RIA/CRISPR-Cas12a detection kit according to claim 7, wherein: the RIA Buffer comprises 70 mM-170 mM of sodium lauroyl sarcosinate, 50mM of Tris-HCl, 20 mM-50 mM of EDTA, 30 mM-75 mM of Triton X-100, 1.2M-3.6M of DMSO and 5 mM-10 mM of DTT.
9. An Asian Zika virus RT-RIA/CRISPR-Cas12a detection method, which is characterized in that the kit of any one of claims 1-8 is adopted, and comprises the following steps:
(1) preparing an RIA reaction system and a Cas12a reaction system;
(2) RIA reaction;
(3) cas12a reaction;
(4) and (6) judging the result.
10. The detection method of Asian Zika virus RT-RIA/CRISPR-Cas12a according to claim 9, comprising the following steps:
(1) preparation of reagents
Preparing a RIA reaction system: preparing 50 mu L of RIA reaction system, wherein 30.0 mu L of RIA Buffer, 10.0 mu L of RIA enzyme and 2.0 mu L of each of upstream and downstream RIA of 10 mu mol/L in the RIA reaction system are added with DEPC water to be supplemented to 45.5 mu L, the mixture is evenly mixed and then placed on ice, 2.0 mu L of serum template is added, and 2.5 mu L of MgOAc RIA activator with the concentration of 280mmol/L is added;
cas12a reaction system was configured: 3 mu L of Cas12a Buffer, 3 mu L of 300nM crRNA, 1 mu M LbCas12a 1 mu L, 1.5 mu L of 10 mu M FQ fluorescent reporter probe, supplementing water to 27 mu L and taking 3 mu L of RIA reaction product as a template;
(2) fluorescence detection
The RIA reaction is carried out on constant temperature equipment or variable temperature PCR equipment, and the reaction conditions are as follows: 15min at 37 ℃; cas12a fluorescence detection is carried out on constant-temperature or variable-temperature real-time fluorescence detection equipment containing FAM or VIC channels, and the reaction conditions are as follows: fluorescence was collected at 37 ℃ for 10min every 0.5 min.
(3) Analysis of results
Quality control standard: the VIC channel is positive, and the fluorescence curve is an obvious S-shaped curve, which indicates that the nucleic acid extraction and amplification reaction is normal;
and (4) interpretation of results: analyzing the result on the premise of qualified quality control, and if an FAM channel has an obvious S-shaped curve or an amplification curve has an exponential growth trend, determining that the result is positive for the Zika virus; otherwise, the result is negative.
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