| It is observed that there is a growing number of people infected with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),along with the impact of corona virus disease 2019(COVID-19)caused by SARS-CoV-2 sweeping over the world since2020.While it has caused countless deaths and economic losses to people worldwide,COVID-19 has made people feel such a kind of social panic and thus evolved as “Public Health Emergency of International Concern”.As SARS-CoV-2 is of strong infectious,mutability and pathogenicity,people are now facing huge challenge to implement outbreak control along with the shortage of treatment and prevention measures to COVID-19.In general,rapid and accurate diagnostic is the first step,which should be taken to prevent the outbreak and can help people detect the distributed cases and screen the key crowds who have close contact with the virus or entry into the country from abroad.As a result,the spreading of COVID-19 can be controlled effectively.Since the outbreak of COVID-19,the launching of nucleic acid detection agents of reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR)and its clinical application have thus played a critical role in diagnosing the patients clinically and screening suspicious patients.However,the traditional nucleic acid detection needs more procedures and requires more time to conduct detection with the limitation of centralized testing.Meanwhile,it also requires professional instrument for testing and professional staffs to conduct detection steps,which cause constraints of time and place of the detection itself.In order to propel the development of COVID-19 diagnostic and break through the current dependence of existing detections on professional instruments,it is of critical need to develop a new nucleic acid detection without professional instrument,thus realizing rapid diagnosis of suspicious patients and close-contact crowd.In recent years,with the rapid development of CRISPR technology,it now has not only been applied to gene editing field but developed with series of CRISPR-based diagnostic.By employing the trans-cleavage activity of CRISPR associated protein(Cas protein)and combing with the isothermal amplification technology,this technology has characteristics of rapid,high sensitivity and high specificity,and can realize the needs of rapid nucleic acid detection outside the laboratory through lateral flow strip,color changes of the solution and so on.As the most common field method for nucleic acid detection,lateral flow strip based on CRISPR will detect the existence of suspicious substances according to the number and location of the bands present with the colloidal gold particles.In a word,it can clearly present the testing results without professional instruments and thus can bring people benefits of rapid,cheap and portable convenience.This research has developed an easy-readout and sensitive enhanced(ERASE)strip based on the fundamentals of CRISPR-Cas13 a nucleic acid detection by combing with the reverse transcription recombinase aided amplification(RT-RAA)and immunochromatography technology.Compared to the original lateral flow strip,this testing paper,which has been made alteration to the concentration of its main components,is of higher sensitivity and can produce results that are more objective.Besides,this research has also developed a field nucleic acid detection for SARS-CoV-2with ERASE strip,in addition,the testing result of 649 clinical samples from 646 patients through ERASE assay and clinical diagnosis as well as the testing result through RT-qPCR assay both have presented good consistency,thus providing a new method for COVID-19 diagnosis in primary areas.1.Development of a "line elimination" lateral flow strip based on CRISPR(ERASE strip)Based on the disadvantages of the sensitivity and objective interpretation in existing lateral flow strip,this research has made alteration to the concentration of the main components within the strip based on RT-RAA isothermal amplification and CRISPR-Cas13 a nucleic acid detection.Meanwhile,it has also screened the reporter RNA preferences,and optimized the portion of the components in CRISPR system and the reaction conditions for each major step.According to the result,while combing the colloidal gold of 0.4 mg/m L,rabbit anti-FITC of 16 ?g/m L and streptavidin of 0.8mg/m L with the reporter RNA of 20 U,it can thus realize to detect specifically(single base)the positive nucleic acid of as low as 1 copy/?L under 37?42? for about 1 hour.Besides,the interpretation criterion of the above strip for a positive result is the test line eliminating absolutely,and it is more sensitive than existing lateral flow strip(10copies/?L).According to the above results,the "line elimination" lateral flow strip based on CRISPR can gain results with more sensitive than that from existing lateral flow strip,with interpretation that is more objective as well.Moreover,this research has also developed a CRISPR nucleic acid detection(ERASE detection)which can produce accurate interpretation with high sensitivity and specificity outside the laboratory.2.Development of an ERASE nucleic acid detection for SARS-CoV-2Based on the needs for conducting rapid nucleic acid detection outside the laboratory,this research has obtained a segment of proper cr RNA target sequence in SARS-CoV-2 N gene by sequence alignment.And this target sequence is of high conservation(with a match ratio of 99.81%)and specificity(discrimination 10 kinds of close coronaviruses);by conducting primer screening,it has also obtained a pair of primers which can high-efficiently amplify the target sequence of cr RNA.Meanwhile,it has also developed an ERASE assay for SARS-CoV-2 nucleic acid detection by employing the cr RNA and RT-RAA primer pair above.In addition,its sensitivity has been evaluated by the standard SARS-CoV-2 RNA of different dilutions.According to the results,the ERASE assay can stably detect SARS-CoV-2 RNA(5/5)of 1 copy/?L,the sensitivity of which approaches that of RT-qPCR assay(0.5 copy/?L).Moreover,its specificity also has been evaluated by other six nucleic acid of human coronavirus and two kinds of coronavirus nucleic acid from the bat.According to the results,there is no cross reaction with other eight coronavirus.Based on the above results,the ERASE assay for SARS-CoV-2 nucleic acid detection is of high sensitivity and specificity and can realize the needs of high sensitivity,specificity and point-of-care testing for SARS-CoV-2.3.Evaluation of SARS-CoV-2 ERASE assay based on clinical samplesIn order to further evaluate the detection effect of SARS-CoV-2 ERASE assay,this research has detected 649 clinical samples from 646 patients in 4 clinical institutions through ERASE assay and RT-qPCR assay,separately.According to the results,the clinical sensitivity of ERASE assay is 91.67% and the clinical specificity is 99.21%,with the clinical overall coincidence of 96.13% compared to clinical diagnosis.And the positive coincidence is 92.02%,the negative coincidence is 99.22% and the overall coincidence is 96.30%,compared to the results of RT-qPCR assay.While among the detection of 53 weakly positive samples,the overall clinical incidence presented by the ERASE assay is 84.91% and the highest value of Ct in positive samples interpreted by ERASE assay is 39.83,which is close to the critical value of RT-qPCR(Ct value ? 40);among the detection of 165 sputum samples,the clinical sensitivity presented by the ERASE assay is 94.67%,which is higher than the value of 90.48% presented while detecting 484 cases of oropharyngeal swabs.Besides,it can detect all the sputum samples with the Ct value lower than 35.Based on the above results,the testing result through ERASE assay and clinical diagnosis as well as the testing result through RT-qPCR assay both have presented good consistency.In a word,this research has developed an ERASE strip applicable to CRISPR based on the lateral flow strip,which intends to improve the sensitivity of CRISPR filed nucleic acid detection and the objectiveness of interpretation as well.Besides,this research has also developed a point-of-care CRISPR nucleic acid detection(ERASE detection)with high objective,sensitivity and specificity,which is interpreted through the results present on ERASE strip by combing the CRISPR-Cas13 a detection with RT-RAA isothermal amplification.Meanwhile,this assay has also been applied to SARS-CoV-2 and the ERASE assay can detect 649 clinical samples from 646 patients in good consistency with clinical diagnosis and RT-qPCR assay,helped nucleic acid detection get rid of the dependence on fluorescence detection equipment,as well as shorten the time and improve the convenience.Besides,it has also provided technological support for the practical implementation of COVID-19 diagnosis and enabled developing countries and primary health agencies to conduct nucleic acid detection rapid to implement. |
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