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1.
Anal Chim Acta ; 1189: 339222, 2022 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-34815046

RESUMO

In this paper preliminarily verified that graphene oxide (GO) nanomaterials enhanced the recombinase polymerase amplification (RPA). GO nanosheets improved the efficiency of RPA amplification by absorbing ingredients to induce local aggregation. The recombinase initially aggregated with the primers to form nucleoprotein filaments, absorbed on the GO nanosheets, changing the structure. Therefore, an isothermal fluorescence biosensor was developed based on GO nanosheets enhanced the RPA to detect RNA interference (RNAi) transgenic plants. FAM-labeled primers were absorbed and quenched by the GO nanosheets. After amplification, the primers were extended into double-stranded DNA, detaching from the GO surface to recover the fluorescent signal. The biosensor displayed high sensitivity and selectivity and showed an excellent relationship ranging from 1.5 to 100 ng of genome DNA, with a detection limit (LOD) of 1.5 ng. Consequently, the biosensor provides an enhanced isothermal method for detecting genetically modified (GM) products and exhibits significant potential for molecular detection.


Assuntos
Técnicas Biossensoriais , Recombinases , Grafite , Técnicas de Amplificação de Ácido Nucleico , Plantas Geneticamente Modificadas/genética , Interferência de RNA
2.
Acta Trop ; 225: 106201, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34688633

RESUMO

Currently utilized molecular detection methods are based mainly on nucleic acid extraction, amplification, and detection procedures that may require costly equipment, numerous reagents, and highly trained personnel. These requirements make diagnostic tests expensive, time-consuming, and not suitable for point-of-care applications. There is an increasing demand for simple, low-cost portable technologies. To overcome these challenges, a paper-based elution independent collection device (EICD) was designed to collect microorganisms and recover nucleic acids for molecular biology applications with minimal steps. In this study, we demonstrate a simpler Anaplasma marginale detection that uses an EICD for nucleic acid collection combined with recombinase polymerase amplification (RPA), and a lateral flow dipstick for detection of the specified target. A pre-lysis blood treatment was optimized that uses Triton X-100 lysis buffer and bovine serum album in wash buffer. Blood samples were incubated for 5 min at room temperature and run through the EICD. Four 1-mm diameter discs excised from EICD were used as template in basic RPA and lateral flow (nfo) (endonuclease IV) RPA assays. Each disc of soluble central membrane (SCM) carried circa 0.249 pg/µl of Anaplasma DNA. The percentage of nucleic acid recoverable from the SCM ranged between 60% - 70%. Blood samples infected with A. marginale were treated with Triton X-100 pre-lysis protocol. All samples tested positive by PCR and RPA methods. EICD-driven collection of blood samples is a practical method successfully adapted to detect Anaplasma spp. or blood-borne pathogen DNA and has potential for point-of-care detection in resource-limited settings.


Assuntos
Anaplasma marginale , Anaplasma , Anaplasma marginale/genética , DNA , Técnicas de Amplificação de Ácido Nucleico , Recombinases , Sensibilidade e Especificidade
3.
World J Gastroenterol ; 27(39): 6631-6646, 2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34754157

RESUMO

BACKGROUND: Different types of pathogenic mutations may produce different clinical phenotypes, but a correlation between Peutz-Jeghers syndrome (PJS) genotype and clinical phenotype has not been found. Not all patients with PJS have detectable mutations of the STK11/LKB1 gene, what is the genetic basis of clinical phenotypic heterogeneity of PJS? Do PJS cases without STK11/LKB1 mutations have other pathogenic genes? Those are clinical problems that perplex doctors. AIM: The aim was to investigate the specific gene mutation of PJS, and the correlation between the genotype and clinical phenotype of PJS. METHODS: A total of 24 patients with PJS admitted to the Air Force Medical Center, PLA (formerly the Air Force General Hospital, PLA) from November 1994 to January 2020 were randomly selected for inclusion in the study. One hundred thirty-nine common hereditary tumor-related genes including STK11/LKB1 were screened and analyzed for pathogenic germline mutations by high-throughput next-generation sequencing (NGS). The mutation status of the genes and their relationship with clinical phenotypes of PJS were explored. RESULTS: Twenty of the 24 PJS patients in this group (83.3%) had STK11/LKB1 gene mutations, 90% of which were pathogenic mutations, and ten had new mutation sites. Pathogenic mutations in exon 7 of STK11/LKB1 gene were significantly lower than in other exons. Truncation mutations are more common in exons 1 and 4 of STK11/LKB1, and their pathogenicity was significantly higher than that of missense mutations. We also found SLX4 gene mutations in PJS patients. CONCLUSION: PJS has a relatively complicated genetic background. Changes in the sites responsible for coding functional proteins in exon 1 and exon 4 of STK11/LKB1 may be one of the main causes of PJS. Mutation of the SLX4 gene may be a cause of genetic heterogeneity in PJS.


Assuntos
Mutação em Linhagem Germinativa , Síndrome de Peutz-Jeghers , Proteínas Serina-Treonina Quinases/genética , Éxons , Humanos , Mutação , Síndrome de Peutz-Jeghers/genética , Fenótipo , Recombinases/genética
4.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 33(5): 452-456, 2021 Oct 27.
Artigo em Chinês | MEDLINE | ID: mdl-34791841

RESUMO

OBJECTIVE: To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania. METHODS: Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 (ITS1) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti, Toxoplasma gondii, Plamodium vivax, P. ovale, P. falciparum, P. malariae, L. donovani and L. infantum. RESULTS: After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 µmol/L and 0.08 µmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum, and no cross-reactions were observed with B. microti, T. gondii, P. vivax, P. ovale, P. falciparum or P. malariae. The lowest limitation of detection of the fluorescent RAA assay was 10 copies/µL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/µL Leishmania genomic DNA. CONCLUSIONS: A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum, which is effective for field screening of leishmaniasis.


Assuntos
Leishmania , Ácidos Nucleicos , Leishmania/genética , Técnicas de Amplificação de Ácido Nucleico , Recombinases , Sensibilidade e Especificidade
5.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 33(5): 464-469, 2021 Oct 26.
Artigo em Chinês | MEDLINE | ID: mdl-34791843

RESUMO

OBJECTIVE: To establish a nucleic acid assay for detection of Paragonimus skrjabini based on the recombinase-aided isothermal amplification (RAA) technique, and to preliminarily evaluate its detection efficiency. METHODS: The metacercariae of P. skrjabini, P. westermani and Euparagonimus cenocopiosus were isolated from crabs, and genomic DNA was extracted for molecular characterization. The cytochrome coxidase 1 (cox1) gene sequence of P. skrjabini was selected as the target gene fragment, and the primers and probes were designed, screened and synthesized for RAA assay. The genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province were used as templates for verification of the fluorescent RAA assay. The fluorescent RAA assay was performed to detect different concentrations of plasmids containing target gene fragment and P. skrjabini metacercariae genomic DNA to determine the sensitivity. Fluorescent RAA assay was performed with recombinant plasmids containing P. skrjabini cox1 gene sequences at different concentrations and P. skrjabini genomic DNA as templates to evaluate its sensitivity, and the genomic DNA of P. westermani, E. cenocopiosus, Clonorchis sinensis and Schistosoma japonicum was detected with fluorescent RAA assay to evaluate its specificity. RESULTS: P. skrjabini, P. westermani and E. cenocopiosus metacercariae were isolated from crabs, respectively. Molecular characterization and phylogenetic analysis confirmed their homology with the genes sequences of standard Paragonimus strains in GenBank. A fluorescent RAA assay was successfully established for nucleic acid detection of P. skrjabini, and the genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province was amplified using the fluorescent RAA assay within 5 min, while the negative control was not amplified. If the recombinant plasmid containing P. skrjabini cox1 gene sequences was used as templates, the fluorescent RAA assay showed the lowest detection limit of 10 copies/µL, and positive amplification was observed within 5 min. If genomic DNA was used as templates, the fluorescent RAA assay showed the lowest detection limit of 10 pg/µL, and all positive amplifications were found within 5 to 10 min. In addition, the fluorescent RAA assay was tested negative for P. westermani, E. cenocopiosus, C. sinensis and S. japonicum. CONCLUSIONS: A rapid, sensitive and specific fluorescent RAA assay is successfully established for nucleic acid detection of P. skrjabini, which has potential values in rapid field detection and species identification in freshwater crabs in areas endemic for P. skrjabini.


Assuntos
Ácidos Nucleicos , Recombinases , Animais , Técnicas de Amplificação de Ácido Nucleico , Filogenia , Recombinases/genética , Sensibilidade e Especificidade
6.
Front Cell Infect Microbiol ; 11: 698929, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34595129

RESUMO

Pseudomonas aeruginosa is a common opportunistic pathogen that causes acute nosocomial necrotizing pneumonia and is the predominant source of chronic lung infections in patients with the genetic disorder cystic fibrosis. Early diagnosis in infected patients and monitoring P. aeruginosa contamination is therefore of great importance in controlling disease spread and development with timely drugs intervention treatment and cut off infection source. Traditional culture-biochemical methods are time consuming and highly dependent on technicians and expensive instruments. To address these challenges, the present study aimed to develop a rapid, sensitive, and specific, on-site detection method for P. aeruginosa based on recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) technology. The experimental process included screening and modification of primer and probe sets targeting the unique virulence gene elastase B (lasB); specificity detection in 29 strains of P. aeruginosa and 23 closely-related pathogenic bacteria; sensitivity measurements with gradient-diluted P. aeruginosa genomic DNA and probit regression analysis; and clinical application evaluation using 574 patients samples and calculating coincidence rate and kappa index value in comparison with the culture-biochemical method. The P. aeruginosa RPA-LFS assay could complete the amplification process at 37°C constant temperature within 30 min and results could be visualized by the naked eye within 10 min on LFS. The assay displayed high sensitivity with a limit of detection of 3.05 CFU/reaction. It also demonstrated high specificity by showing no cross reaction with other pathogenic bacteria, and rapidness by being completed in less than an hour. Furthermore, when used with clinical samples, the assay had a coincidence rate of 98.26% with the culture-biochemical method and a kappa index value of 0.9433. These data indicate that the RPA-LFS assay represents a major improvement for P. aeruginosa detection, especially in resource-limited areas.


Assuntos
Pseudomonas aeruginosa , Recombinases , Humanos , Técnicas de Amplificação de Ácido Nucleico , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Recombinases/genética , Sensibilidade e Especificidade , Tecnologia
7.
Front Cell Infect Microbiol ; 11: 746325, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34616692

RESUMO

Klebsiella pneumoniae carbapenemase genes (bla KPC) play an important role in carbapenem-resistant Enterobacteriaceae in China. A rapid detection method for bla KPC genes and investigations into the molecular characteristics of bla KPC positive Klebsiella pneumoniae were necessary. In this study, an easy and rapid recombinase aided amplification assay (RAA) for bla KPC was established. This protocol could be completed at 39°C in 15-20 min. The sensitivity of this assay was determined as 48 copies per reaction, and the specificity was 100%. The bla KPC RAA method could be used for clinical diagnosis and epidemiological investigation. Among 801 fecal samples from inpatients, 34 bla KPC positive isolates were identified from each sample, of which 23 isolates were K. pneumoniae. ST11 with bla KPC-2 was the most prevalent type. All these strains were multidrug resistant and carried various virulence genes. Fecal carriage of bla KPC positive carbapenem-resistant K.pneumoniae poses significant challenges for public health control.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Humanos , Infecções por Klebsiella/diagnóstico , Klebsiella pneumoniae/genética , Recombinases/genética , beta-Lactamases/genética
8.
ACS Sens ; 6(10): 3773-3780, 2021 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-34595928

RESUMO

Isothermal amplification reactions represent an important and exciting approach to achieve widespread, low cost, and easily implemented molecular diagnostics. This work presents a modified recombinase polymerase amplification (RPA) reaction, which can be directly coupled to a simple electrochemical measurement to ultimately allow development of a nucleic acid-based assay for antibiotic resistance genes. It is shown that use of reagents from a standard RPA reaction kit allows incorporation of horse radish peroxidase-labeled thymine nucleotides into amplified DNA strands, which can be detected via an amperometric signal readout for detection of important gene sequences. The assay is exemplified through detection of fragments of the oxacillin resistance gene in Escherichia coli cells bearing a drug resistance plasmid, achieving a potential limit of detection of 319 cfus/mL and an unoptimized time to result of 60 min. This work serves as a suitable demonstration of the potential for a system to deliver detection of key drug resistance genes at clinically relevant levels.


Assuntos
Técnicas de Amplificação de Ácido Nucleico , Oxacilina , Escherichia coli/genética , Peroxidase do Rábano Silvestre , Recombinases
9.
J Cell Sci ; 134(20)2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34515305

RESUMO

The advent of modern single-cell biology has revealed the striking molecular diversity of cell populations once thought to be more homogeneous. This newly appreciated complexity has made intersectional genetic approaches essential to understanding and probing cellular heterogeneity at the functional level. Here, we build on previous knowledge to develop a simple adeno-associated virus (AAV)-based approach to define specific subpopulations of cells by Boolean exclusion logic (AND NOT). This expression by Boolean exclusion (ExBoX) system encodes for a gene of interest that is turned on by a particular recombinase (Cre or FlpO) and turned off by another. ExBoX allows for the specific transcription of a gene of interest in cells expressing only the activating recombinase, but not in cells expressing both. We show the ability of the ExBoX system to tightly regulate expression of fluorescent reporters in vitro and in vivo, and further demonstrate the adaptability of the system by achieving expression of a variety of virally delivered coding sequences in the mouse brain. This simple strategy will expand the molecular toolkit available for cell- and time-specific gene expression in a variety of systems.


Assuntos
Neurônios , Recombinases , Animais , Expressão Gênica , Camundongos , Recombinases/genética
10.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 33(4): 334-338, 2021 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-34505438

RESUMO

OBJECTIVE: To develop a rapid test for detection of Schistosoma japonicum specific gene fragments based on the recombinase-aided isothermal amplification assay (RAA) and nucleic acid dipstick test. METHODS: The S. japonicum SjG28 gene fragment was selected as the target gene fragment, and the primers and fluorescent probe were designed and synthesized. Then, a S. japonicum nucleic acid dipstick test was established. The sensitivity of this dipstick test was evaluated by detecting different copies of recombinant plasmids containing the S. japonicum SjG28 gene fragment and different concentrations of genomic DNA from adult worms of S. japonicum, and the specificity of the dipstick test was evaluated by detecting the genomic DNA from Clonorchis sinensis, S. mansoni, Ancylostoma duodenale, S. haematobium, Babesia and Paragonimus westermani. RESULTS: The S. japonicum nucleic acid dipstick test based on the S. japonicum SjG28 gene fragment showed the minimum detectable limit of 10 copies/µL of the recombinant plasmid containing the S. japonicum SjG28 gene fragment and the minimum detectable limit of 1 pg/µL of S. japonicum genomic DNA, and the dipstick assay tested negative for the genomic DNA from C. sinensis, S. mansoni, A. duodenale, S. haematobium, Babesia and P. westermani. CONCLUSIONS: A rapid, simple, and visualized assay is established for detection of S. japonicum specific gene fragments based on RAA and nucleic acid dipstick test.


Assuntos
Ácidos Nucleicos , Schistosoma japonicum , Animais , Técnicas de Amplificação de Ácido Nucleico , Recombinases , Schistosoma japonicum/genética , Sensibilidade e Especificidade
11.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 33(4): 339-345, 2021 Aug 19.
Artigo em Chinês | MEDLINE | ID: mdl-34505439

RESUMO

OBJECTIVE: To establish a multiplex nucleic acid assay for rapid detection of Echinococcus multilocularis, E. granulosus and E. shiquicus based on the recombinase-aided isothermal amplification assay (RAA) and to preliminarily assess its diagnostic efficiency. METHODS: The mitochondrial genomic sequences of E. multilocularis (GenBank accession number: NC_000928), E. granulosus (GenBank accession number: NC_044548) and E. shiquicus (GenBank accession number: NC_009460) were used as target sequences, and three pairs of primers were designed based on the RAA primer design principle and synthesized for the subsequent multiple RAA amplification. The genomic DNA of E. multilocularis, E. granulosus and E. shiquicus at different concentrations and the recombinant plasmids containing the target gene at various concentrations were amplified to evaluate the diagnostic sensitivity of the multiplex RAA assay, and the genomic DNA of E. multilocularis, E. granulosus, E. shiquicus, Taenia multiceps, T. saginata, T. asiatica, Dipylidium caninum, T. hydatigena, Toxocara canis, Fasciola hepatica, T. pisiformis, Mesocestoides lineatus and Cryptosporidiumn canis was detected using the multiplex RAA assay to evaluate its specificity. In addition, the reaction condition of the multiplex RAA assay was optimized, and was then employed to detect the tissues with echinococcosis lesions, simulated canine fecal samples and field captured fox fecal samples to examine its application values. RESULTS: The multiplex RAA assay was effective to specifically amplify the mitochondrial gene fragments of E. multilocularis, E. granulosus and E. shiquicus within 40 min at 39 °C, with sequence lengths of 540, 430 bp and 200 bp, respectively. This multiplex RAA assay showed the minimum detection limits of 2.0, 2.5 pg/µL and 3.1 pg/µL for detection of the genomic DNA of E. multilocularis, E. granulosus and E. shiquicus, and presented the minimum detection limit of 200 copies/µL for detection of the recombinant plasmids containing E. multilocularis, E. granulosus and E. shiquicus target genes. This multiplex RAA assay was effective to simultaneously detect single and multiple infections with E. multilocularis, E. granulosus and E. shiquicus, but failed to amplify the genomic DNA of T. multiceps, T. saginata, T. asiatica, D. caninum, T. hydatigena, T. canis, F. hepatica, T. pisiformis, M. lineatus and C. canis. In addition, the optimized multiplex RAA assay was effective to detect all positive samples from the tissue samples with echinococcosis lesions, simulated canine fecal samples and field captured fox fecal samples, which was fully consistent with the detection of the single PCR assay. CONCLUSIONS: A sensitive and specific multiplex nucleic acid assay for rapid detection of E. multilocularis, E. granulosus and E. shiquicus has been successfully established.


Assuntos
Equinococose , Echinococcus multilocularis , Animais , Cães/parasitologia , Equinococose/diagnóstico , Equinococose/veterinária , Echinococcus multilocularis/isolamento & purificação , Raposas/parasitologia , Ácidos Nucleicos , Recombinases , Sensibilidade e Especificidade
12.
Biomed Environ Sci ; 34(8): 650-655, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34474727

RESUMO

Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two of the most prevalent human herpesviruses, cause a wide spectrum of diseases and symptoms and are associated with serious health problem. In this study, we developed an internal control reference recombinase-aided amplification (ICR-RAA) assay for the rapid detection of EBV and CMV within 30 min. The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV, respectively, with no cross reaction with other pathogens. In comparison with those of the commercial quantitative polymerase chain reaction (qPCR), the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33% and 84.84%, respectively; the specificity was 98.75% and 100.00%, respectively; and the Kappa values were 0.930 and 0.892 ( P < 0.05), respectively. In comparison with those of qPCR, the sensitivity of the EBV and CMV ICR-RAAs using the DNA by thermal lysis was 72.22% and 80.00%, respectively; the specificity was 100.00%; and the Kappa values were 0.764 and 0.878 ( P < 0. 05), respectively. Thus, rapid and specific detection of EBV and CMV is possible using ICR-RAA assays.


Assuntos
Citomegalovirus/genética , DNA Viral/análise , Herpesvirus Humano 4/genética , Técnicas de Amplificação de Ácido Nucleico , Recombinases/genética , Adolescente , Adulto , Criança , Pré-Escolar , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/virologia , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/virologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Adulto Jovem
13.
Anal Chem ; 93(37): 12808-12816, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34506127

RESUMO

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.


Assuntos
COVID-19 , Transcrição Reversa , Teste para COVID-19 , Humanos , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , Recombinases/genética , SARS-CoV-2 , Sensibilidade e Especificidade , Tecnologia
14.
Anal Chem ; 93(40): 13641-13650, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34586776

RESUMO

A multiplex assay based on recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a desirable tool for many areas. This multiplex assay could be efficiently realized using single-stranded (ss) DNAs located in separate zones on the test strip and bound complementary ssDNA tags of double-stranded (ds) DNA amplicons. Here, we investigate how to enrich multiplex assay capabilities using ssDNAs. Bifunctional oligonucleotide probes integrating (1) a forward primer for RPA, (2) a C9 spacer to stop polymerase, and (3) a ssDNA tag for binding at test strip are developed. The amplicons have a unique individual ssDNA tag at one end and a universal label of fluorescein introducing through a reverse primer at the other end. A conjugate of gold nanoparticles (GNP) with antibodies to fluorescein is used to detect all amplicons. The remainder of primers after RPA interacting with GNP conjugate was found to be a limiting factor for sensitive and specific multiplex assay. The addition of anti-RPA-primers before the use of test strips was proposed to simply and effectively eliminate remaining primers. This approach was successfully applied for the detection of three priority plant RNA viruses: potato virus Y (PVY), -S (PVS) and potato leafroll virus (PLRV). The total time of the assay is 30 min. The multiplex RPA-LFT detected at least 4 ng of PVY per g of plant leaves, 0.04 ng/g for PVS, and 0.04 ng/g for PLRV. The testing of healthy and infected potato samples showed concordance between the developed assay and reverse transcription-polymerase chain reaction. Thus, the capabilities of the proposed universal modules (ssDNA anchors, bifunctional probes, and blocking anti-primers) for multiplex detection of RNA analytes with high specificity and sensitivity were demonstrated.


Assuntos
Nanopartículas Metálicas , Vírus de Plantas , Primers do DNA , Ouro , Técnicas de Amplificação de Ácido Nucleico , Recombinases , Sensibilidade e Especificidade
15.
Yi Chuan ; 43(8): 802-812, 2021 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-34413019

RESUMO

The genetically modified (GM) maize 'Shuangkang'12-5 has good insect resistance and herbicide tolerance, which is one of the first series of GM maizes obtained a safety certificate in China, and it has broad application prospect in the future. This study established an on-site rapid detection method for GM 'Shuangkang'12-5 based on recombinase polymerase amplification (RPA) technology, which primes and probe were designed according to the specific flank sequence. Then the best combination of primers and probe was obtained through a screeing process. The amplification results of fluorescence RPA can be directly visualized under blue light. The results showed that the visual detection system of GM 'Shuangkang'12-5 with high specificity, and the detection sensitivity of the method could reached 10 copies. Further research found that the RPA amplification system had a wide range of temperature (34℃-46℃). According to this property, the common self-heating warm pastes on the market were used replace the traditional heating instruments to stimulate the RPA.The results showed that the self-heating warm paste meets the temperature requirement of the RPA system. Finally, we combined the self-heating warm pastes with the RPA visual detection system to conduct on-site detection of GM 'Shuangkang'12-5, and compared the results with the detection results of qPCR. The detection showed that the results of on-site visual detection method established in this study were consistent with the detection results of the qPCR. Moreover, the visual detection method was more shorter in time and the final detection result was clear and easy to distinguish. The rapid on-site visual detection method for GM 'Shuangkang' 12-5 established in this study has high specificity, high sensitivity and convenience. It not only meets the needs of on-site rapid detection of GM 'Shuangkang'12-5, but also provides highlight for the development of other on-site rapid detection methods.


Assuntos
Recombinases , Zea mays , Primers do DNA , Técnicas de Amplificação de Ácido Nucleico , Nucleotidiltransferases , Zea mays/genética
16.
Analyst ; 146(17): 5271-5279, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34355716

RESUMO

The ability to visually detect low numbers of Salmonella in food samples is highly valuable but remains a challenge. Here we present a novel platform for ultrasensitive and visual detection of Salmonella Typhimurium by integrating the bio-barcode immunoassay (BCA), recombinase polymerase amplification (RPA), and CRISPR-Cas12a cleavage in a single reaction system (termed as BCA-RPA-Cas12a). In the system, the target bacteria were separated by immunomagnetic nanoparticles and labeled with numerous barcode AuNPs, which carry abundant bio-barcode DNA molecules to amplify the signal. Afterwards, the bio-barcode DNA molecules were amplified by RPA and subsequently triggered the cleavage activity of Cas12a to generate the fluorescence signal. Due to this triplex signal amplification, the BCA-RPA-Cas12a system can selectively detect Salmonella Typhimurium at the single-digit level with the naked eye under blue light within 60 min. Meanwhile, this novel platform was successfully applied to detect Salmonella Typhimurium in spiked milk samples with a similar sensitivity and satisfactory recovery, indicating its potential application in real samples. Furthermore, in virtue of the versatility of the antibody in the stage of BCA, the BCA-RPA-Cas12a system can be extended to further application in other bacteria detection and food safety monitoring.


Assuntos
Nanopartículas Metálicas , Recombinases , Sistemas CRISPR-Cas , Ouro , Imunoensaio , Técnicas de Amplificação de Ácido Nucleico , Recombinases/genética , Salmonella typhimurium/genética
17.
J Biomed Nanotechnol ; 17(7): 1364-1370, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34446139

RESUMO

Researchers have conducted in-depth research on DNA methylation mechanism, which is related to various diseases such as deficiency of imprinted gene and occurrence of tumors. This study provides a novel rapid quantitative detection assay and real-time fluorescence recombinase-aided amplification assay (RAA) for DNA methylation. Firstly, specific sequence of methylation genes was chosen and primers and fluorogenic probe for RAA experiment were designed and synthesized. Lastly, these amplification products were proven by sequencing and analysis. Results showed that the amplification efficiency and template concentration of RAA had linear dependent (R² > 95%) when the concentration range was 4.64×108 copies/µL˜4.64×104 copies/µL. The test assay can also detect positive samples when the template concentration is below 4.64×104 copies/µL. Remarkably, the entire experiment process only takes 15-20 minutes, so it is beneficial for rapid bedside simple screening of some special DNA methylation sites, such as detection of resistance genes. In a word, this method has very great potential for diseases with DNA methylation in clinical settings, especially if methylation analysis needs to be done quickly and easily.


Assuntos
Metilação de DNA , Recombinases , Primers do DNA , Técnicas de Amplificação de Ácido Nucleico , Recombinases/genética , Recombinases/metabolismo , Sensibilidade e Especificidade
18.
Sci Rep ; 11(1): 15962, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354122

RESUMO

Anaplasma marginale, A. ovis, and A. phagocytophilum are the causative agents of bovine anaplasmosis, ovine anaplasmosis, and granulocytic anaplasmosis, respectively. The gold standard for diagnosis of post-acute and long-term persistent infections is the serological cELISA, which does not discriminate between Anaplasma species and requires highly equipped laboratories and trained personnel. This study addresses the development of a rapid, isothermal, sensitive, species-specific RPA assays to detect three Anaplasma species in blood and cELISA A. marginale-positive serum samples. Three RPA primer and probe sets were designed targeting msp4 genes of each Anaplasma species and the internal control (GAPDH gene) for each assay. The limit of detection of gel-based or RPA-basic assays is 8.99 × 104 copies/µl = A. marginale, 5.04 × 106 copies/µl = A. ovis, and 4.58 × 103 copies/µl = A. phagocytophilum, and for each multiplex lateral flow or RPA-nfo assays is 8.99 × 103 copies/µl of A. marginale, 5.04 × 103 copies/µl of A. ovis, 4.58 × 103 copies/µl of A. phagocytophilum, and 5.51 × 103 copies/µl of internal control (GAPDH). Although none of the 80 blood samples collected from Oklahoma cattle were positive, the RPA-nfo assays detected all A. marginale cattle blood samples with varying prevalence rates of infection, 83% of the 24 cELISA A. marginale-positive serum samples, and all A. phagocytophilum cell culture samples. Overall, although early detection of three Anaplasma species was not specifically addressed, the described RPA technique represents an improvement for detection of three Anaplasma in regions where access to laboratory equipment is limited.


Assuntos
Anaplasma/genética , Anaplasmose/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Anaplasma/isolamento & purificação , Anaplasma/patogenicidade , Anaplasma marginale/genética , Anaplasma ovis/genética , Anaplasma phagocytophilum/genética , Anaplasmose/genética , Anaplasmose/microbiologia , Animais , Bovinos , DNA Bacteriano/genética , Limite de Detecção , Recombinases/metabolismo
19.
BMC Vet Res ; 17(1): 286, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433470

RESUMO

BACKGROUND: Epizootic haemorrhagic disease virus (EHDV) and the Palyam serogroup viruses (PALV) have led to significant economic losses associated with livestock production globally. A rapid, sensitive and specific method for the detection of EHDV and PALV is critical for virus detection, monitoring, and successful control and elimination of related diseases. RESULTS: In the present study, a recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) assay for the co-detection of genome segment 1 (Seg-1) of EHDV and PALV was developed and evaluated. The analytical sensitivities of the established RPA-LFD assay in the detection of EHDV and PALV were 7.1 copies/µL and 6.8 copies/µL, respectively. No cross-reaction with other members of the genus Orbivirus, including African horse sickness virus, bluetongue virus, Guangxi orbivirus, Tibet orbivirus and Yunnan orbivirus was observed. The established RPA-LFD assay accurately detected 39 EHDV strains belonging to 5 serotypes and 29 PALV strains belonging to 3 serotypes. The trace back results of quantitative real-time polymerase chain reaction (qRT-PCR) and the established RPA-LFD assay on sentinel cattle were consistent. The coincidence rates of qRT-PCR and the established RPA-LFD assay in 56 blood samples from which EHDV or PALV had been isolated and 96 blood samples collected from cattle farms were more than 94.8 %. The results demonstrated that the established RPR-LFD assay is specific, sensitive and reliable, and could be applied in early clinical diagnosis of EHDV and PALV. CONCLUSIONS: This study highlights the development and application of the RPA-LFD assay in the co-detection of EHDV and PALV for the first time. The assay could be used as a potential optional rapid, reliable, sensitive and low-cost method for field diagnosis of EHDV and PALV.


Assuntos
Vírus da Doença Hemorrágica Epizoótica/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/veterinária , Vírus Palyam/isolamento & purificação , Testes Sorológicos/veterinária , Animais , Bioensaio/veterinária , Bovinos , Vírus da Doença Hemorrágica Epizoótica/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírus Palyam/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Recombinases , Infecções por Reoviridae/diagnóstico , Infecções por Reoviridae/veterinária , Sensibilidade e Especificidade , Sorogrupo , Testes Sorológicos/métodos
20.
Commun Biol ; 4(1): 875, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267310

RESUMO

Many synthetic gene circuits are restricted to single-use applications or require iterative refinement for incorporation into complex systems. One example is the recombinase-based digitizer circuit, which has been used to improve weak or leaky biological signals. Here we present a workflow to quantitatively define digitizer performance and predict responses to different input signals. Using a combination of signal-to-noise ratio (SNR), area under a receiver operating characteristic curve (AUC), and fold change (FC), we evaluate three small-molecule inducible digitizer designs demonstrating FC up to 508x and SNR up to 3.77 dB. To study their behavior further and improve modularity, we develop a mixed phenotypic/mechanistic model capable of predicting digitizer configurations that amplify a synNotch cell-to-cell communication signal (Δ SNR up to 2.8 dB). We hope the metrics and modeling approaches here will facilitate incorporation of these digitizers into other systems while providing an improved workflow for gene circuit characterization.


Assuntos
Engenharia Genética/métodos , Recombinases/genética , Transdução de Sinais , Biologia Sintética/métodos , Curva ROC
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