Novel degenerate primer sets for the detection and identification of emaraviruses reveal new chrysanthemum species

AbstractOver the course of the COVID-19 pandemic, several SARS-CoV-2 genetic variants of concern have appeared and spread throughout the world. Detection and identification of these variants is important to understanding and controlling their rapid spread. Current detection methods for a particularly concerning variant, B.1.1.7, require expensive qPCR machines and depend on the absence of a signal rather than a positive indicator of variant presence. Here we report an assay using a pair of molecular beacons paired with reverse transcription loop mediated amplification to allow isothermal amplification from saliva to specifically detect B.1.1.7 and other variants which contain a characteristic deletion in the gene encoding the viral spike protein. This assay is specific, affordable and allows multiplexing with other SARS-CoV-2 LAMP primer sets.

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Comparison of Molecular Procedures for Detection and Identification of Guignardia citricarpa and G. mangiferae

Plant Disease ◽

10.1094/pdis-91-5-0525 ◽

2007 ◽

Vol 91 (5) ◽

pp. 525-531 ◽

Cited By ~ 34

Author(s):

N. A. Peres ◽

R. Harakava ◽

G. C. Carroll ◽

J. E. Adaskaveg ◽

L. W. Timmer

Keyword(s):

Extraction Procedure ◽

Citrus Fruit ◽

Black Spot ◽

Polymerase Chain Reaction Primer ◽

Internal Transcribed Spacer Region ◽

Guignardia Citricarpa ◽

Detection And Identification ◽

Specificity And Sensitivity ◽

Fruit Spot ◽

Primer Sets

Citrus black spot, caused by Guignardia citricarpa, is a serious fruit spot disease and is widely distributed in Asia, southern Africa, and South America, but does not occur in North America or the Mediterranean region. A nonpathogenic species, G. mangiferae, is cosmopolitan with a wide host range and can colonize citrus fruit and leaves saprophytically. Detection and identification of Guignardia spp. on citrus fruit is necessary for epidemiological, management, and regulatory purposes. In this study, we compared published and unpublished polymerase chain reaction primer sets for their specificity and sensitivity in the detection and differentiation of the two Guignardia spp. All primers evaluated successfully identified the two species using purified DNA from fungal cultures or mycelia as source materials. However, some primer sets were not highly effective in detecting G. citricarpa when DNA was extracted directly from single characteristic black spot lesions on fruit. Thus, new primer pairs for both species were designed from the internal transcribed spacer region that were highly sensitive and specific for detection of G. citricarpa using DNA recovered from single lesions on fruit by a rapid DNA extraction procedure.

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Detection and identification of azithromycin resistance mutations on Treponema pallidum 23S rRNA gene by nested multiplex polymerase chain reaction

Medical Journal of Indonesia ◽

10.13181/mji.v26i2.1543 ◽

2017 ◽

Vol 26 (2) ◽

pp. 90-6 ◽

Cited By ~ 1

Author(s):

Desy A. Gultom ◽

Yeva Rosana ◽

Ida Efendi ◽

Wresti Indriatmi ◽

Andi Yasmon

Keyword(s):

Multiplex Polymerase Chain Reaction ◽

Treponema Pallidum ◽

23S Rrna ◽

Rrna Gene ◽

Chain Reaction ◽

Detection And Identification ◽

23S Rrna Gene ◽

Polymerase Chain ◽

Primer Sets ◽

Blood Specimens

Background: Azithromycin-resistant strains of Treponema pallidum is associated with the mutation of 23S rRNA gene of T. pallidum. Although these strains are now prevalent in many countries, there is no laboratory test kit to detect and identify these mutations. Thus, in this study we developed a nested multiplex polymerase chain reaction (PCR) to detect and identify A2058G and A2059G mutations in 23S rRNA gene.Methods: Three primer sets were designed for nested PCR reactions. To obtain maximum PCR reaction, all parameters were optimized. The specificity of the primer sets was evaluated towards some microorganisms. A sensitivity test was conducted to get detection limit of deoxyribonucleic acid (DNA). Forty-five whole blood specimens were tested by PCR, and positive results were confirmed by the DNA sequencing.Results: The assay could detect at least 4,400 DNA copy number and showed no cross reaction with other microorganisms used in the specificity test. A total 13 of 45 whole blood specimens were PCR positive for T. pallidum, and no single mutations (either A2058G or A2059G) were detected. Two positive specimens were confirmed by the DNA sequencing and showed no mutation.Conclusion: Nested multiplex PCR developed in this study showed a specific and sensitive test for the detection and identification of A2058G and/or A2059G mutations of 23S rRNA T. pallidum gene.

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De-MetaST-BLAST: A Tool for the Validation of Degenerate Primer Sets and Data Mining of Publicly Available Metagenomes

PLoS ONE ◽

10.1371/journal.pone.0050362 ◽

2012 ◽

Vol 7 (11) ◽

pp. e50362 ◽

Cited By ~ 8

Author(s):

Christopher A. Gulvik ◽

T. Chad Effler ◽

Steven W. Wilhelm ◽

Alison Buchan

Keyword(s):

Data Mining ◽

Degenerate Primer ◽

Primer Sets

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PCR-based assays for the fish pathogen Aeromonas salmonicida. I. Evaluation of three PCR primer sets for detection and identification

Diseases of Aquatic Organisms ◽

10.3354/dao049129 ◽

2002 ◽

Vol 49 ◽

pp. 129-138 ◽

Cited By ~ 20

Author(s):

HK Byers ◽

N Gudkovs ◽

MSJ Crane

Keyword(s):

Aeromonas Salmonicida ◽

Fish Pathogen ◽

Detection And Identification ◽

Primer Sets ◽

Pcr Primer

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Method for Rapid Detection and Identification of Chaetomium and Evaluation of Resistance to Peracetic Acid

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-543 ◽

2013 ◽

Vol 76 (6) ◽

pp. 999-1005 ◽

Cited By ~ 11

Author(s):

MOTOKAZU NAKAYAMA ◽

KOUICHI HOSOYA ◽

DAISUKE TOMIYAMA ◽

TAKASHI TSUGUKUNI ◽

TETSUHIRO MATSUZAWA ◽

...

Keyword(s):

Peracetic Acid ◽

Cell Walls ◽

Acid Resistance ◽

High Specificity ◽

Specific Primers ◽

Beverage Industry ◽

Detection And Identification ◽

Transmission Electron ◽

Pcr Products ◽

Primer Sets

In the beverage industry, peracetic acid has been increasingly used as a disinfectant for the filling machinery and environment due to merits of leaving no residue, it is safe for humans, and its antiseptic effect against fungi and endospores of bacteria. Recently, Chaetomium globosum and Chaetomium funicola were reported resistant to peracetic acid; however, little is known concerning the detail of peracetic acid resistance. Therefore, we assessed the peracetic acid resistance of the species of Chaetomium and related genera under identical conditions and made a thorough observation of the microstructure of their ascospores by transmission electron microscopy. The results of analyses revealed that C. globosum and C. funicola showed the high resistance to peracetic acid (a 1-D antiseptic effect after 900 s and 3-D antiseptic effect after 900 s) and had thick cell walls of ascospores that can impede the action mechanism of peracetic acid. We also developed specific primers to detect the C. globosum clade and identify C. funicola by using PCR to amplify the β-tubulin gene. PCR with the primer sets designed for C. globosum (Chae 4F/4R) and C. funicola (Cfu 2F/2R) amplified PCR products specific for the C. globosum clade and C. funicola, respectively. PCR with these two primer sets did not detect other fungi involved in food spoilage and environmental contamination. This detection and identification method is rapid and simple, with extremely high specificity.

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Identification of hepatitis a virus in cell cultures by solid phase immune electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142815 ◽

1986 ◽

Vol 44 ◽

pp. 238-239

Author(s):

C.D. Humphrey ◽

T.L. Cromeans ◽

E.H. Cook ◽

D.W. Bradley

Keyword(s):

Electron Microscopy ◽

Liquid Phase ◽

Cell Cultures ◽

Rapid Detection ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Solid Phase ◽

Immune Electron Microscopy ◽

Detection And Identification

There is a variety of methods available for the rapid detection and identification of viruses by electron microscopy as described in several reviews. The predominant techniques are classified as direct electron microscopy (DEM), immune electron microscopy (IEM), liquid phase immune electron microscopy (LPIEM) and solid phase immune electron microscopy (SPIEM). Each technique has inherent strengths and weaknesses. However, in recent years, the most progress for identifying viruses has been realized by the utilization of SPIEM.

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