Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification

Objective To establish an ultra-sensitive,ultra-fast,visible detection method for Vibrio parahaemolyticus(VP).Methods We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a(CRISPR/Cas12a)combined with recombinase polymerase amplification and visual detection(CRISPR/Cas12a-VD).Results CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10^(-18)M(single molecule detection)within 30 min without cross-reactivity against other bacteria.When detecting pure cultures of VP,the consistency of results reached 100%compared with real-time PCR.The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10^(2)CFU/g.Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods,such as real-time PCR,and has great potential for preventing the spread of pathogens.

Establishment of Recombinase Polymerase Amplification for Rapid Detection of Spring Viremia of Carp Virus(SVCV)

[Objective]The paper was to develop a rapid method for the detection of spring Viremia of carp virus(SVCV).[Method]The specific primers were designed by targeting the G gene of SVCV.The recombinase polymerase amplification(RPA)assay for detecting SVCV was estab-lished by optimizing the reaction conditions.The optimal amplification temperature of RPA assay was 30℃,and the test could be finished within 20 min.[Result]The method was specific with no cross-reaction with other common fish infectious viruses.Sensitivity test showed that the lowest detection limit of the method was 89.2 copies/μL,higher than that of traditional RT-PCR.Moreover,a total of 80 clinical samples were detected by RPA and RT-PCR,respectively.The weak positive samples tested by RT-PCR could be detectable with RPA,indicating that RPA assay could be used in clinical detection.[Conclusion]The method established is rapid,simple,specific and sensitive for testing SVCV,and it will be widely used in grassroots laboratory and on-site inspection.

Establishment of a rapid detection method of Ureaplasma urealyticum based on recombinant polymerase amplification

Ureaplasma urealyticum(UU),is one of the most vital pathogens causing genitourinary tract infections of the body,and it can result in poor maternal and perinatal outcomes.The aim of this study was to establish a method to detect Ureaplasma urealyticum based on recombinant polymerase amplification(RPA)technique.Specific primers and probes were designed according to the 16sRNA gene sequence of Ureaplasma urealyticum.Six pathogens were detected for real-time fluorescence RPA specificity verification,including Mycoplasma hominis(MH),Chlamydia trachomatis(CT),Neisseria gonorrhoeae(NG),Staphylococcus aureus,Escherichia coli,and Lactobacillus vaginalis.The sensitivity of the method was performed by gradient dilution of the extracted template.A total of 60 clinical samples were detected by the established real-time fluorescence RPA.Detection of Ureaplasma urealyticum can be completed within 20 minutes at 39°C using established RPA method.The minimum detection limit of Ureaplasma urealyticum by real-time fluorescence RPA was 3 pg.The evaluation of 60 clinical samples proved that RPA method was feasible.A high specificity,sensitivity,simplicity and rapidity method for Ureaplasma urealyticum detection was successfully established based on the real-time fluorescence RPA method.

Research progress and prospects of nucleic acid isothermal amplification technology

Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.

A dual-RPA based lateral flow strip for sensitive,on-site detection of CP4-EPSPS and Cry1Ab/Ac genes in genetically modified crops

Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient.In this study,a one-tube dual recombinase polymerase amplification(RPA)reaction system for CP4-EPSPS and Cry1Ab/Ac was proposed and combined with a lateral flow immunochromatographic assay,named“Dual-RPA-LFD”,to visualize the dual detection of genetically modified(GM)crops.In which,the herbicide tolerance gene CP4-EPSPS and the insect resistance gene Cry1Ab/Ac were selected as targets taking into account the current status of the most widespread application of insect resistance and herbicide tolerance traits and their stacked traits.Gradient diluted plasmids,transgenic standards,and actual samples were used as templates to conduct sensitivity,specificity,and practicality assays,respectively.The constructed method achieved the visual detection of plasmid at levels as low as 100 copies,demonstrating its high sensitivity.In addition,good applicability to transgenic samples was observed,with no cross-interference between two test lines and no influence from other genes.In conclusion,this strategy achieved the expected purpose of simultaneous detection of the two popular targets in GM crops within 20 min at 37°C in a rapid,equipmentfree field manner,providing a new alternative for rapid screening for transgenic assays in the field.

Development of RPA-Cas12a-fluorescence assay for rapid and reliable detection of human bocavirus 1

Human bocavirus(HBoV)1 is considered an important pathogen that mainly affects infants aged 6–24 months,but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of individuals with early infection of HBoV1 remains somewhat challenging.Herein,we present a novel faster,lower cost,reliable method for the detection of HBoV1,which integrates a recombinase polymerase amplification(RPA)assay with the CRISPR/Cas12a system,designated the RPA-Cas12a-fluorescence assay.The RPA-Cas12a-fluorescence system can specifically detect target gene levels as low as 0.5 copies of HBoV1 plasmid DNA per microliter within 40 min at 37℃without the need for sophisticated instruments.The method also demonstrates excellent specificity without cross-reactivity to non-target pathogens.Furthermore,the method was appraised using 28 clinical samples,and displayed high accuracy with positive and negative predictive agreement of 90.9%and 100%,respectively.Therefore,our proposed rapid and sensitive HBoV1 detection method,the RPA-Cas12a-fluorescence assay,shows promising potential for early on-site diagnosis of HBoV1 infection in the fields of public health and health care.The established RPA-Cas12a-fluorescence assay is rapid and reliable method for human bocavirus 1 detection.The RPA-Cas12a-fluorescence assay can be completed within 40 min with robust specificity and sensitivity of 0.5 copies/μl.

A Microfluidic System with Active Mixing for Improved Real-Time Isothermal Amplification

To improve the performance of real-time recombinase polymerase amplification(RPA),a microfluidic system with active mixing is developed to optimize the reaction dynamics.Instead of adopting a single typical reaction chamber,a specific reactor including a relatively large chamber in center with two adjacent zig-zag channels at two sides is integrated into the microfluidic chip.Active mixing is achieved by driving the viscous reagent between the chamber and the channel back and forth periodically with an outside compact peristaltic pump.To avoid reagent evapora-tion,one end of the reactor is sealed with paraffin oil.A hand-held companion device is developed to facilitate real-time RPA amplification within 20 min.The whole area of the reactor is heated with a resistance heater to provide uniform reaction temperature.To achieve real-time monitoring,a compact fluorescence detection module is integrated into the hand-held device.A smartphone with custom application software is adopted to control the hand-held device and display the real-time fluorescence curves.The performances of two cases with and without active on-chip mixing are compared between each other by detecting African swine fever viruses.It has been demonstrated that,with active on-chip mixing,the amplification efficiency and detection sensitivity can be signifi-cantly improved.

Programmable endonuclease combined with isothermal polymerase amplification to selectively enrich for rare mutant allele fractions

Liquid biopsy is a highly promising method for non-invasive detection of tumor-associated nucleic acid fragments in body fluids but is challenged by the low abundance of nucleic acids of clinical interest and their sequence homology with the vast background of nucleic acids from healthy cells.Recently,programmable endonucleases such as clustered regularly interspaced short palindromic repeats(CRISPR)associated protein(Cas)and prokaryotic Argonautes have been successfully used to remove background nucleic acids and enrich mutant allele fractions,enabling their detection with deep next generation sequencing(NGS).However,the enrichment level achievable with these assays is limited by futile binding events and off-target cleavage.To overcome these shortcomings,we conceived a new assay(Programmable Enzyme-Assisted Selective Exponential Amplification,PASEA)that combines the cleavage of wild type alleles with concurrent polymerase amplification.While PASEA increases the numbers of both wild type and mutant alleles,the numbers of mutant alleles increase at much greater rates,allowing PASEA to achieve an unprecedented level of selective enrichment of targeted alleles.By combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification,we converted samples with0.01%somatic mutant allele fractions(MAFs)to products with 70%MAFs in a single step within 20 min,enabling inexpensive,rapid genotyping with such as Sanger sequencers.Furthermore,PASEA's extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA probes.Real-time PASEA'performance was proved equivalent to clinical amplification refractory mutation system(ARMS)-PCR and NGS when testing over hundred cancer patients'samples.This strategy has the potential to reduce the cost and time of cancer screening and genotyping,and to enable targeted therapies in resource-limited settings.

RPA-Assisted Cas12a System for Detecting Pathogenic Xanthomonas oryzae,a Causative Agent for Bacterial Leaf Blight Disease in Rice

Xanthomonas oryzae pv.oryzae(Xoo)is a widespread pathogen causing bacterial leaf blight(BLB)disease,devastating rice productivity in many cultivated areas of Thailand.A specific and simple method for Xoo detection is required to improve surveillance of disease transmission and outbreak.This study developed a recombinase polymerase amplification(RPA)assay assisted with CRISPR-cas12a assay(RAC)for Xoo detection from bacterial cell suspension of infected rice samples without DNA extraction.The efficiency of the RAC system for Xoo detection using either Xoo80 or Xoo4009 locus was optimized to amplify and determine the sensitivity and specificity using a Xoo DNA template from bacterial cell suspension of infected rice samples without DNA extraction.The RAC system using the Xoo4009 locus gave a higher specificity than Xoo80 locus,because only Xoo species was amplified positive RPA product with fluorescence signal by cas12a digestion,which indicated no cross reactivity.Optimal RAC using the Xoo4009 locus enabled diagnosis of Xoo presence from both plant extracted samples of Xoo artificially inoculated rice leaves within 3 d post-inoculation without symptomatic BLB appearance,and Xoo naturally infected rice.Findings exhibited that RAC using the Xoo4009 locus offered sensitivity,specificity and simplicity for Xoo detection,with low intensities of Xoo-DNA(1×10^(3) copies/μL)and Xoo-cell(2.5×10^(3) cfu/mL).This developed RAC system showed significantly potential for Xoo detection at point-of-care application for early signs of BLB disease outbreak in rice fields.

Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in Acinetobacter baumannii

Background:Acinetobacter baumannii(A.baumannii)is one of the pivotal pathogens responsible for nosocomial infections,especially in patients with low immune response,and infection with carbapenem-resistant A.baumannii has been increasing in recent years.Rapid and accurate detection of carbapenem-resistance genes in A.baumannii could be of immense help to clinical staff.Methods:In this study,a 15-μL reaction system for recombinase polymerase amplification(RPA)was developed and tested.We collected 30 clinical isolates of A.baumannii from the Burn Institute of Southwest Hospital of Third Military Medical University(Army Medical University)for 6 months and tested antibiotic susceptibility using the VITEK 2 system.A.baumannii was detected based on the blaOXA-51 gene by PCR,qPCR and 15μL-RPA,respectively.Sensitivity and specificity were evaluated.In addition,PCR and 15μL-RPA data for detecting the carbapenem-resistance gene blaOXA-23 were comparatively assessed.Results:The detection limit of the blaOXA-51 gene by 15μL RPA was 2.86 CFU/ml,with sensitivity comparable to PCR and qPCR.No positive amplification signals were detected in non-Acinetobacter isolates,indicating high specificity.However,only 18 minutes were needed for the 15μL RPA assay.Furthermore,an antibiotic susceptibility test showed that up to 90%of A.baumannii strains were resistant to meropenem and imipenem;15μL RPA data for detecting blaOXA-23 showed that only 10%(n=3)of A.baumannii isolates did not show positive amplification signals,and the other 90%of(n=27)isolates were positive,corroborating PCR results.Conclusion:We demonstrated that the new 15μL RPA assay for detecting blaOXA-23 in A.baumannii is faster and simpler than qPCR and PCR.It is a promising alternative molecular diagnostic tool for rapid and effective detection of A.baumannii and drug-resistance genes in the field and point-ofcare testing.

Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques

African swine fever(ASF)is an infectious disease caused by African swine fever virus(ASFV)with clinical symptoms of high fever,hemorrhages and high mortality rate,posing a threat to the global swine industry and food security.Quarantine and control of ASFV is crucial for preventing swine industry from ASFV infection.In this study,a recombinase polymerase amplification(RPA)-CRISPR-based nucleic acid detection method was developed for diagnosing ASF.As a highly sensitive method,RPA-CRISPR can detect even a single copy of ASFV plasmid and genomic DNA by determining fluorescence signal induced by collateral cleavage of CRISPR-lw Cas13 a(previously known as C2c2)through quantitative real-time PCR(q PCR)and has the same or even higher sensitivity than the traditional q PCR method.A lateral flow strip was developed and used in combination with RPA-CRISPR for ASFV detection with the same level of sensitivity of Taq Man q PCR.Likewise,RPA-CRISPR is capable of distinguishing ASFV genomic DNA from viral DNA/RNA of other porcine viruses without any cross-reactivity.This diagnostic method is also available for diagnosing ASFV clinical DNA samples with coincidence rate of 100%for both ASFV positive and negative samples.RPA-CRISPR has great potential for clinical quarantine of ASFV in swine industry and food security.