Rapid nucleic acid detection of Zaire ebolavirus on paper fluidics

The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV−2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV−2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus.

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Highly Specific and Sensitive Detection of Yersinia pestis by Portable Cas12a-UPTLFA Platform

Frontiers in Microbiology ◽

10.3389/fmicb.2021.700016 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yang You ◽

Pingping Zhang ◽

Gengshan Wu ◽

Yafang Tan ◽

Yong Zhao ◽

...

Keyword(s):

Nucleic Acid ◽

Yersinia Pestis ◽

Rapid Detection ◽

Detection Method ◽

Detection System ◽

Point Of Care ◽

High Sensitivity ◽

Nucleic Acid Detection ◽

Great Promise ◽

High Background

The recent discovery of collateral cleavage activity of class-II clustered regularly interspaced short palindromic repeats–CRISPR-associated protein (CRISPR-Cas) makes CRISPR-based diagnosis a potential high-accuracy nucleic acid detection method. Colloidal gold-based lateral flow immunochromatographic assay (LFA), which has been combined with CRISPR/Cas-based nucleic detection, usually associates with drawbacks of relative high background and the subjectivity in naked-eye read-out of the results. Here, we developed a novel system composed of Cas12a-based nucleic acid detection and up-converting phosphor technology (UPT)-based LFA (UPT–LFA), termed Cas12a-UPTLFA. We further demonstrated the utility of this platform in highly sensitive and specific detection of Yersinia pestis, the causative agent of the deadly plague. Due to high infectivity and mortality, as well as the potential to be misused as bioterrorism agent, a culture-free, ultrasensitive, specific, and rapid detection method for Y. pestis has long been desired. By incorporating isothermal recombinase polymerase amplification, the Cas12a-UPTLFA we established can successfully detect genomic DNA of Y. pestis as low as 3 attomolar (aM) and exhibited high sensitivity (93.75%) and specificity (90.63%) for detection of spiked blood samples with a detection limit of 102 colony-forming unit per 100 μl of mouse blood. With a portable biosensor, Cas12a-UPTLFA assay can be operated easily by non-professional personnel. Taken together, we have developed a novel Cas12a-UPTLFA platform for rapid detection of Y. pestis with high sensitivity and specificity, which is portable, not expensive, and easy to operate as a point-of-care method. This detection system can easily be extended to detect other pathogens and holds great promise for on-site detection of emerging infectious pathogens.

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Rapid Detection of Bacillus cereus Using Cross-Priming Amplification

Journal of Food Protection ◽

10.4315/0362-028x.jfp-19-156 ◽

2019 ◽

Vol 82 (10) ◽

pp. 1744-1750 ◽

Cited By ~ 1

Author(s):

JING ZHANG ◽

BIAO DI ◽

HONGBO SHAN ◽

JUNHUA LIU ◽

YONG ZHOU ◽

...

Keyword(s):

Detection Limit ◽

Nucleic Acid ◽

Bacillus Cereus ◽

Rapid Detection ◽

Genomic Dna ◽

Food Samples ◽

Nucleic Acid Detection ◽

Practical Applications ◽

Gastrointestinal Pathogen ◽

Life Threatening

ABSTRACT Bacillus cereus is a spore-forming gastrointestinal pathogen that can cause life-threatening diseases. Here, a simple and effective assay to detect B. cereus was developed, using cross-priming amplification (CPA). Amplicons were detected using disposable cartridges that contained nucleic acid detection strips. The sensitivity of CPA assay for B. cereus was assessed using serial dilutions of genomic DNA, which indicated a detection limit of 3.6 × 101 CFU/mL. No cross-reactions were detected when genomic DNA extracted from 12 different B. cereus strains and 20 other bacterial foodborne strains were tested, suggesting that the assay is highly specific. Finally, we evaluated the practical applications of the CPA assay for the detection of B. cereus in 150 food samples and found that its sensitivity and specificity, compared with real-time PCR, were approximately 98.18 and 100%, respectively. In conclusion, CPA combined with nucleic acid detection strips is easy to perform, requires simple equipment, and offers highly specific and sensitive B. cereus detection.

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Rapid Detection of Avian Leukosis Virus Subgroup J by Cross-priming Amplification

10.21203/rs.2.21696/v1 ◽

2020 ◽

Author(s):

Yong Xiang ◽

Lizhen Li ◽

Peng Liu ◽

Ling Yan ◽

Zeng Jiang ◽

...

Keyword(s):

Nucleic Acid ◽

Rapid Detection ◽

Dna Amplification ◽

High Specificity ◽

Avian Leukosis Virus ◽

Nucleic Acid Detection ◽

Poultry Production ◽

Aerosol Pollution ◽

Subgroup J ◽

Avian Leukosis Virus Subgroup

Abstract Avian leukosis virus subgroup J (ALV-J) causes oncogenic disease in chickens in China, resulting in great harm to poultry production. Herein, we employed a cross-priming amplification (CPA) approach and a nucleic acid detection device to establish a visual rapid detection method for ALV-J. When the amplification reaction was carried out at 60°C for just 60 min, the sensitivity of CPA was 10 times higher than conventional PCR, with high specificity, based on detection of 123 clinical plasma samples. The coincidence rate with real-time PCR was 97.3% (71/73). CPA detection of ALV-J does not require an expensive PCR instrument; a simple water bath or incubator is sufficient for complete DNA amplification, and the closed nucleic acid detection device avoids aerosol pollution, making judgment of results more intuitive and objective. The CPA assay would be a promising simple, rapid and sensitive method for identification of ALV-J.

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Rapid detection of avian leukosis virus subgroup J by cross-priming amplification

Scientific Reports ◽

10.1038/s41598-021-90479-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yong Xiang ◽

Lizhen Li ◽

Peng Liu ◽

Ling Yan ◽

Zeng Jiang ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Cell Cultures ◽

Real Time Pcr ◽

Rapid Detection ◽

Avian Leukosis Virus ◽

Nucleic Acid Detection ◽

Rt Pcr ◽

Subgroup J ◽

Avian Leukosis Virus Subgroup

AbstractAvian leukosis virus subgroup J (ALV-J) causes oncogenic disease in chickens in China, resulting in great harm to poultry production, and remains widespread in China. Herein, we employed a cross-priming amplification (CPA) approach and a nucleic acid detection device to establish a visual rapid detection method for ALV-J. The sensitivity of CPA, polymerase chain reaction (PCR) and real-time PCR (RT-PCR) was compared, and the three methods were used to detect ALV-J in the cell cultures which inoculated with clinical plasma. The result showed when the amplification reaction was carried out at 60 °C for just 60 min, the sensitivity of CPA was 10 times higher than conventional PCR, with high specificity, which was comparable with RT-PCR, based on detection of 123 cell cultures which inoculated with clinical plasma, the coincidence rate with real-time PCR was 97.3% (71/73). CPA detection of ALV-J does not require an expensive PCR instrument; a simple water bath or incubator is sufficient for complete DNA amplification, and the closed nucleic acid detection device avoids aerosol pollution, making judgment of results more intuitive and objective. The CPA assay would be a promising simple, rapid and sensitive method for identification of ALV-J.

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