A Sensitive, Highly Specific Novel Isothermal Amplification Method Based on Single-Nucleotide Polymorphism for the Rapid Detection of Salmonella Pullorum

Shen, Huiyong; Wen, Jing; Liao, Xinmeng; Lin, Quankui; Zhang, Jianfeng; Chen, Kaifeng; Wang, Shaojun; Zhang, Jianmin

doi:10.3389/fmicb.2020.560791

Cited by 22 publications

(16 citation statements)

References 21 publications

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“…In the CP-LAMP ASFV detection method, based on the original primer sets, we targeted the 9GL gene sequence by designing a new fluorophore quencher-labeled cleaved probe with a ribonucleotide insertion; RNase H2 is only activated when the probe perfectly matches the mutant target, leading to the hydrolytic release of a quencher moiety, and consequently an amplified signal (Shen et al, 2020). Our CP-LAMP reaction can be measured in real time using a simple thermocycler to quantify fluorescence; it does not require any additional fluorescent intercalating dyes.…”

Section: Discussionmentioning

confidence: 99%

“…Based on our previous report (Shen et al, 2020), we successfully set up a basic reaction system using standard plasmids. The reaction system contained 2.5 mL of buffer (10×), 1.5 mL of MgSO 4 , 4 mL of dNTPs, 8 U/µL of Bst 2.0 WarmStart DNA polymerase (1 µL), 0.1 U/µL of RNase H2 Enzyme (0.3 µL), 0.3 µL of probe (10 µM), 4 µL of FIP/BIP primer (10 µM), 0.5 µL of F3/B3 primer (10 µM), 1.5 µL of loop primer (10 µM), and 2.5 µL of DNA sample.…”

Section: Primer Design Optimization and Establishment Of The Basic Re...mentioning

confidence: 99%

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Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus

Wang

Shen

Lin

et al. 2022

Front. Cell. Infect. Microbiol.

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African Swine Fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and lethal viral disease of pigs. However, commercial vaccines are not yet available, and neither are drugs to prevent or control ASF. Therefore, rapid, accurate on-site diagnosis is urgently needed for detection during the early stages of ASFV infection. Herein, a cleaved probe-based loop-mediated isothermal amplification (CP-LAMP) detection method was established. Based on the original primer sets, we targeted the ASFV 9GL gene sequence to design a probe harboring a ribonucleotide insertion. Ribonuclease H2 (RNase H2) enzyme activity can only be activated when the probe is perfectly complementary, resulting in hydrolytic release of a quencher moiety, and consequent signal amplification. The method displayed robust sensitivity, with copy number detection as low as 13 copies/µL within 40 min at constant temperature (62°C). Visualization of the fluorescence product was employed using a self-designed 3D-printed visualization function cassette, and the CP-LAMP method achieved specific identification and visual detection of ASFV. Moreover, coupling the dual function cassette and smartphone quantitation makes the CP-LAMP assay first user-friendly, cost-effective, portable, rapid, and accurate point-of-care testing (POCT) platform for ASFV.

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Section: Discussionmentioning

confidence: 99%

Section: Primer Design Optimization and Establishment Of The Basic Re...mentioning

confidence: 99%

Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus

Wang

Shen

Lin

et al. 2022

Front. Cell. Infect. Microbiol.

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“…The CP-RT-LAMP basic reaction system was established as described previously (18) and effectively improved as follows: the system included 2.…”

Section: Establishment and Optimization Of The Basic Reaction Systemmentioning

confidence: 99%

A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus

et al. 2022

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Porcine deltacoronavirus (PDCoV) causes watery diarrhea, vomiting, and 30–40% mortality in newborn piglets. A simple, rapid, and sensitive method for PDCoV detection is valuable in its surveillance and control. Here, we developed a novel, cleaved probe-based reverse transcription loop-mediated isothermal amplification (CP-RT-LAMP) method for PDCoV detection. A cleaved probe with a ribonucleotide insertion that targeted the N gene of PDCoV was designed. During the reaction, the enzyme ribonuclease H2 is activated only when the cleaved probe is perfectly complementary to the template, leading to the hydrolytic release of a quencher moiety and signal output. This method can be easily used on a real-time fluorescence quantitative equipment or an on-site isothermal instrument combined with a smartphone. The specificity assay showed no cross-reactivity with other porcine enteric pathogens. This method had a detection limit of 25 copies/μL, suggesting comparable sensitivity with reverse transcription quantitative PCR (RT-qPCR). In detecting 100 clinical samples (48 fecal swab specimens and 52 intestinal specimens), the detection rate of the CP-RT-LAMP method (26%) was higher than that of RT-qPCR (17%). Thus, it is a highly specific and sensitive diagnostic method for PDCoV, with a great application potential for monitoring PDCoV in the laboratory or point-of-care testing in the field.

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“…Much like PCR, isothermal PCR uses enzymatic amplification to amplify a nucleic acid sequence with a polymerase, but isothermal nucleic acid amplification does not require variable temperature cycling. These methods are beginning to provide the sensitivity and specificity needed for detecting single nucleotide changes (Zhou et al 2018a;Shen et al 2020). One recent innovation is to couple the amplification power of isothermal DNA polymerases with CRISPR-Cas specificity (Kellner et al 2019).…”

Section: Isothermal Dna Detection and Crispr-cas-mediated Edit Detectionmentioning

confidence: 99%

Detection of genome edits in plants—from editing to seed

Shillito

Whitt

Ross

et al. 2021

In Vitro Cell.Dev.Biol.-Plant

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Genome editing (also known as gene editing) employs a range of tools such as Meganucleases, Zinc Finger Nucleases, TALENs, and more recently CRISPR to make defined changes in genes, regulatory sequences, untranslated regions, or intergenic regions. It is increasingly being applied in plant science research and to improve plant varieties. The benefits of having effective detection tools begin with optimization of the genome editing process itself and continue with selection and characterization of tissue cultures and/or regenerated plants. Detection tools are also used throughout the breeding process, and for preparation of regulatory dossiers when required, as well as for seed production, and may be necessary for monitoring products in the marketplace. Detection and identification of genome edits employs a wide range of analytical approaches including PCR, digital PCR, and sequencing methods. This article examines the applicability of each category of detection or identification approach, from the optimization of genome editing processes, through creation of edits, selection and characterization, and breeding. The challenges surrounding the detection of genome edits present at low levels in large seed, plant, or grain populations and of differentiating directed genome edits from conventional mutations are also explained.

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A Sensitive, Highly Specific Novel Isothermal Amplification Method Based on Single-Nucleotide Polymorphism for the Rapid Detection of Salmonella Pullorum

Cited by 22 publications

References 21 publications

Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus

Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus

A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus

Detection of genome edits in plants—from editing to seed

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