Dual-target one-step nested PCR for sensitive detection of SARS-CoV-2 nucleic acids

Citrus Huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of its causal agent, Candidatus Liberibacter asiaticus (CLas), is critical for early diagnosis and successful management of HLB. However, current nucleic acid-based detection methods are often insufficient for the early detection of CLas from asymptomatic tissue, and unsuitable for high-throughput and field-deployable diagnosis of HLB. Here we report the development of the Cas12a-based DETECTR (DNA endonuclease-targeted CRISPR trans reporter) assay for highly specific and sensitive detection of CLas nucleic acids from infected samples. The DETECTR assay, which targets the five-copy nrdB gene specific to CLas, couples isothermal amplification with Cas12a trans-cleavage of fluorescent reporter oligos and enables detection of CLas nucleic acids at the attomolar level. The DETECTR assay was capable of specifically detecting the presence of CLas across different infected citrus, periwinkle and psyllid samples, and shown to be compatible with lateral flow assay technology for potential field-deployable diagnosis. The improvements in detection sensitivity and flexibility of the DETECTR technology position the assay as a potentially suitable tool for early detection of CLas in infected regions.

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One-Step Modification of Electrode Surface for Ultrasensitive and Highly Selective Detection of Nucleic Acids with Practical Applications

Analytical Chemistry ◽

10.1021/acs.analchem.6b01250 ◽

2016 ◽

Vol 88 (15) ◽

pp. 7583-7590 ◽

Cited By ~ 21

Author(s):

Chao Li ◽

Dan Wu ◽

Xiaolu Hu ◽

Yang Xiang ◽

Yongqian Shu ◽

...

Keyword(s):

Nucleic Acids ◽

Electrode Surface ◽

Selective Detection ◽

Practical Applications ◽

One Step

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A room-temperature adenosine-based molecular beacon for highly sensitive detection of nucleic acids

Chemical Communications ◽

10.1039/c2cc31382k ◽

2012 ◽

Vol 48 (50) ◽

pp. 6262 ◽

Cited By ~ 24

Author(s):

Yen-Hsiu Lin ◽

Wei-Lung Tseng

Keyword(s):

Nucleic Acids ◽

Room Temperature ◽

Molecular Beacon ◽

Sensitive Detection ◽

Highly Sensitive ◽

Highly Sensitive Detection

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Improved ionic-liquid-functionalized macroporous supports able to purify nucleic acids in one step

Materials Today Bio ◽

10.1016/j.mtbio.2020.100086 ◽

2020 ◽

Vol 8 ◽

pp. 100086

Author(s):

M.C. Neves ◽

P. Pereira ◽

A.Q. Pedro ◽

J.C. Martins ◽

T. Trindade ◽

...

Keyword(s):

Ionic Liquid ◽

Nucleic Acids ◽

One Step

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In SituReverse Transcriptase-Nested Polymerase Chain Reaction to Identify Intracellular Nucleic Acids without the Necessity of Dnase Pretreatment and Hybridisation

Analytical Cellular Pathology ◽

10.1155/2001/654016 ◽

2001 ◽

Vol 22 (3) ◽

pp. 151-158 ◽

Cited By ~ 1

Author(s):

Mario Menschikowski ◽

Margot Vogel ◽

Rolf Eckey ◽

Gerd Dinnebier ◽

Werner Jaross

Keyword(s):

Polymerase Chain Reaction ◽

Nucleic Acids ◽

Nested Pcr ◽

In Situ Hybridisation ◽

Target Sequence ◽

Chain Reaction ◽

Multiple Control ◽

Nested Polymerase Chain Reaction ◽

Polymerase Chain

In the present study a protocol of in situ reverse transcriptase‐nested polymerase chain reaction (in situ RT‐nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by “DNA repair mechanisms” and “endogenous priming”, a two‐step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin‐labeled nucleotides and intrinsic primers. (ii) In order to prevent amplifications of genomic DNA nested primer pairs were applied crossing intron sequences. (iii) To minimize the diffusion of PCR products in cells, the extrinsic primers were extended with complementary 5′‐tails. This approach results in the generation of high molecular weight concatamers during PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT‐nested PCR, which in comparison to the method of in situ RT‐PCR‐in situ‐hybridisation is simpler and less time‐consuming, can be used as an alternative approach to identify intracellular nucleic acids.

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Detection of Mycoplasma hyopneumoniae by Air Sampling with a Nested PCR Assay

Applied and Environmental Microbiology ◽

10.1128/aem.64.2.543-548.1998 ◽

1998 ◽

Vol 64 (2) ◽

pp. 543-548 ◽

Cited By ~ 78

Author(s):

Katharina D. C. Stärk ◽

Jacques Nicolet ◽

Joachim Frey

Keyword(s):

Nested Pcr ◽

Gene Segment ◽

Mycoplasma Hyopneumoniae ◽

Field Conditions ◽

Pcr Analysis ◽

Direct Extraction ◽

Pcr Assay ◽

Sampling System ◽

Respiratory Problems ◽

One Step

ABSTRACT This article describes the first successful detection of airborneMycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 104 times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity.

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