Ligation-Independent Mechanism of Multiplex Ligation-Dependent Probe Amplification

Uno, Naoki; Yanagihara, Katsunori

doi:10.2116/analsci.30.805

Cited by 3 publications

(3 citation statements)

References 14 publications

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“…targets with a single primer pair Thomas et al., 2010a;Uno and Yanagihara, 2014). This system provides several advantages compared to real-time PCR.…”

Section: Insect Sample Preparation and Pathogen Scanningmentioning

confidence: 99%

Simultaneous detection of fungal, bacterial, and viral pathogens in insects by multiplex PCR and capillary electrophoresis

Kwak¹,

Nam²,

Choi³

et al. 2015

International Journal of Industrial Entomology

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Beetles Protaetia brevitarsis seulensis Kolbe (Coleoptera: Cetoniidae) and Allomyrina dichotoma Linn. (Coleoptera: Scarabaeidae) are widely used in traditional medicine, and the number of insect-rearing farms is increasing in South Korea. The purpose of this study was to establish a multiplex PCR-based assay for rapid simultaneous detection of multiple pathogens causing insect diseases. Six insect parasites such as fungi Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) and Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae), bacteria Bacillus thuringiensis Berliner (Bacillales: Bacillaceae), Pseudomonas aeruginosa Migula (Pseudomonadales: Pseudomonadaceae), and Serratia marcescens Bizio (Enterobacteriales: Enterobacteriaceae), and Oryctes rhinoceros nudivirus were chosen based on the severity and incidence rate of insect diseases in South Korea. Pathogen-specific primers were designed and successfully applied for simultaneous detection of multiple infectious agents in farm-bred insects P. b. seulensis and A. dichotoma using multiplex PCR and high resolution capillary electrophoresis. Our results indicate that multiplex PCR is an effective and time-saving method for simultaneous detection of multiple infections in insects, and the QIAxcel capillary electrophoresis system is useful for quantitative evaluation of the individual impact of each infectious agent on the severity of insect disease. The approach designed in this study can be utilized for rapid and accurate diagnostics of infection in insect farms.© 2015 The Korean Society of Sericultural Sciences Int. J. Indust. Entomol. 30(2), 64-74 (2015) IntroductionFollowing a global trend of using natural products from insects, the Korean government has been encouraging the development of insect resources for environmental and dietary purposes, traditional medicine, and as pets (Kim et al., 2013 Vol. 30, No. (1), pp. 64-74 (2015) Pseudomonas aeruginosa is reported as a virulent bacterial pathogen not only for insects but also for mammals (Jander et al., 2000), where it causes infection of the respiratory tract, especially in cystic fibrosis patients (Spiker et al., 2004). Similar to Pseudomonas aeruginosa, Serratia marcescens is known as an opportunistic bacterium (El-Aasar et al., 2013); it is also used to control apple maggot fly population and as a source of insecticidal agents such as toxin luxA (Hejazi and Falkiner, 1997; Lauzon et al., 2003). Oryctes rhinoceros virus Rhabdionvirus oryctes Hüger was first discovered in oil palms in Malaysia 40 years ago and is currently used for the control of rhinoceros beetle population in Malaysia (Huger, 2005;Ramle et al., 2005).We have identified this virus in an A. dichotoma-breeding farm in 2014 in Korea (Lee, 2015).Extensive large-scale insect breeding has resulted in frequent occurrence of insect infection due to multiple pathogens. Here, we developed a multiplex PCR-based approach that allowed simultaneous detection of six insect pathogens using specific primer se...

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“…targets with a single primer pair Thomas et al., 2010a;Uno and Yanagihara, 2014). This system provides several advantages compared to real-time PCR.…”

Section: Insect Sample Preparation and Pathogen Scanningmentioning

confidence: 99%

Simultaneous detection of fungal, bacterial, and viral pathogens in insects by multiplex PCR and capillary electrophoresis

Kwak¹,

Nam²,

Choi³

et al. 2015

International Journal of Industrial Entomology

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“…We previously uncovered a ligation-independent pathway of MLPA through which products of MLPA could be amplified without hybridization and ligation processes (Uno and Yanagihara, 2014). The ligation-independent pathway led to notable levels of amplification.…”

mentioning

confidence: 99%

“…When the mutation is present, amplification is refractory because of a mismatch between the 3′ end of the left probe and the template. We previously showed that ligation-independent probe amplification could not distinguish the point mutation in oligonucleotide templates (Uno and Yanagihara, 2014), but we tried to increase its fidelity by optimizing PCR conditions. We designed an additional probe pair targeting N-acetylmuramoyl-L-alanine amidase (lytA), which is commonly present in S. pneumoniae (Sheppard et al, 2004), to confirm the presence of the bacterium.…”

mentioning

confidence: 99%

Clinical application of a ligation-independent pathway of multiplex ligation-dependent probe amplification for the determination of quinolone susceptibility of Streptococcus pneumoniae

Uno

Araki

Kaku

et al. 2016

Journal of Microbiological Methods

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Heterogeneous spectrum of EXT gene mutations in Chinese patients with hereditary multiple osteochondromas

Li¹,

Wang²,

Tang

et al. 2018

Medicine

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Hereditary multiple osteochondroma (HMO) is one of the most common genetic skeletal disorders. It is caused by mutations in either EXT1 or EXT2 resulting in abnormal skeletal growth and morphogenesis. However, the spectrum and frequency of EXT1 and EXT2 mutations in Chinese patients with HMO was not previously investigated.Mutations were identified by performing Sanger sequencing analysis of the complete coding regions and flanking intronic sequences of EXT1 and EXT2, followed by multiplex ligation-dependent probe amplification (MLPA) analysis to detect gene deletions or duplications that could not be identified by the Sanger sequencing method.The present study identified pathogenic mutations in 93% (68/73) of unrelated HMO probands from 73 pedigrees. Mutations in EXT1 and EXT2 were identified in 53% (39/73) and 40% (29/73) of families. We identified 58 distinct mutations in EXT1 and EXT2, including 20 frameshift mutations, 16 nonsense mutations, 7 missense mutations, 9 splice site mutations, 5 large deletions, and 1 in-frame deletion mutation. Twenty-six of these mutations were novel and 32 were previously reported. Most of the mutations in EXT1 were base deletions or insertions (21/33), whereas the majority of those in EXT2 were single base substitution (18/25).Complete sequencing of both the EXT1 and EXT2 followed by MLPA analysis is recommended for genetic analysis of Chinese patients with HMO. This study provides a comprehensive characterization of the genetic aberrations found in Chinese patients with HMO and highlights the diagnostic value of molecular genetic analysis in this particular disease.

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Ligation-Independent Mechanism of Multiplex Ligation-Dependent Probe Amplification

Cited by 3 publications

References 14 publications

Simultaneous detection of fungal, bacterial, and viral pathogens in insects by multiplex PCR and capillary electrophoresis

Simultaneous detection of fungal, bacterial, and viral pathogens in insects by multiplex PCR and capillary electrophoresis

Clinical application of a ligation-independent pathway of multiplex ligation-dependent probe amplification for the determination of quinolone susceptibility of Streptococcus pneumoniae

Heterogeneous spectrum of EXT gene mutations in Chinese patients with hereditary multiple osteochondromas

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