scholarly journals Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum

Biomédica ◽  
2018 ◽  
Vol 38 (1) ◽  
pp. 111 ◽  
Author(s):  
Rubén E. Varela-M ◽  
Jinney Stefany Arias ◽  
Luz Elena Velásquez

Introducción. En el mundo, las angiostrongilosis de mayor impacto en salud humana y animal son ocasionadas por Angiostrongylus cantonensis, A. costaricensis y A. vasorum. En las personas, las formas clínicas son la meningitis eosinofílica y la angiostrongilosis abdominal, y, en los mamíferos cánidos, el daño cardiopulmonar. Se las consideran enfermedades emergentes debido a la propagación mundial del caracol africano Lissachatina fulica, un huésped intermediario de los parásitos. Los escasos métodos de identificación de Angiostrongylus spp. no son muy específicos ni sensibles y son costosos. Se necesita urgentemente una herramienta diagnóstica asequible, sensible y específicapara el manejo de las angiostrongilosis humana y la animal.Objetivo. Desarrollar una prueba de PCR múltiple en tiempo real (qPCR) para identificar las tres especies patógenas de Angiostrongylus.Materiales y métodos. Mediante un análisis bioinformático se seleccionó una secuencia del genoma ITS-2 de Angiostrongylus para garantizar la especificidad del cebador y las sondas. El ADN de los parásitos adultos (control positivo) y de las larvas se extrajo con el estuche DNeasyBlood & Tissue®.Las reacciones de la PCR cuantitativa se ejecutaron en un termociclador Smartcycler Cepheid®, usando el estuche de mezcla maestra QuantiTect®. Como control negativo, se utilizó ADN humano, de otros parásitos y del caracol africano.Resultados. Los valores del ciclo umbral para los controles positivos de ADN fueron: 21 para Angiostrongylus cantonensis, 22 para A. costaricensis y 31 para A. vasorum. En los controles negativos, el ciclo umbral fue cero. La qPCR mostró una eficiencia de amplificación de 2 (100 %).Conclusiones. En el laboratorio se estandarizó una qPCR múltiple para tres especies clínicamente significativas de Angiostrongylus.

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Andrew Jenkins ◽  
Cecilie Raasok ◽  
Benedikte N. Pedersen ◽  
Kristine Jensen ◽  
Åshild Andreassen ◽  
...  

After publication of our article [1] it came to our notice that the source of the sequence for the control plasmid, pNeo (Materials and methods: Controls) was incorrectly stated as AB094461. The correct accession number is AB074461. The authors apologize for any confusion this may have caused.


2009 ◽  
Vol 9 (1) ◽  
Author(s):  
Martina Wernecke ◽  
Ciara Mullen ◽  
Vimla Sharma ◽  
John Morrison ◽  
Thomas Barry ◽  
...  

2010 ◽  
Vol 16 (6) ◽  
pp. 774-779 ◽  
Author(s):  
F. Wallet ◽  
S. Nseir ◽  
L. Baumann ◽  
S. Herwegh ◽  
B. Sendid ◽  
...  

2016 ◽  
Vol 82 ◽  
pp. S31
Author(s):  
A. Jassem ◽  
M. Krajden ◽  
D. Getman ◽  
P. Hovey ◽  
C. Hentzen ◽  
...  

2007 ◽  
Vol 227 (3) ◽  
pp. 789-798 ◽  
Author(s):  
Maher Chaouachi ◽  
Marie Noelle Fortabat ◽  
Angèle Geldreich ◽  
Pierre Yot ◽  
Camille Kerlan ◽  
...  

2009 ◽  
Vol 85 (4) ◽  
pp. 264-269 ◽  
Author(s):  
A Gayet-Ageron ◽  
B Ninet ◽  
L Toutous-Trellu ◽  
S Lautenschlager ◽  
H Furrer ◽  
...  

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 250
Author(s):  
Kazuki Fujiwara ◽  
Kenta Tomimura ◽  
Toru Iwanami ◽  
Takashi Fujikawa

Background: ‘Candidatus Liberibacter asiaticus’ (CLas) is a major causal agent of citrus greening disease. The disease primarily involves an asymptomatic, often latent infection of CLas. However, there is no effective technique to distinguish latent-infected trees from healthy ones. This study describes the development of a new detection method for latent CLas infection using cuttings. Methods: Root tissues regenerated from cuttings using symptomatic and asymptomatic citrus trees were prepared for real-time a polymerase chain reaction (PCR) test which was used to investigate latent CLas. When some of the regenerated roots were negative for CLas in the first real-time PCR assay, a subsequent cultivation in soils was performed using the CLas-negative cuttings. CLas development during cultivation was evaluated by a second real-time PCR assay using soil-grown roots from seedlings. Results: Previously, CLas had not been detected from leaves of the latent-infected trees in our greenhouse by real-time PCR. In this study, however, CLas was detected at a moderate frequency from the root tissues of cuttings derived from the latent-infected trees, by the same PCR test. For cuttings with regenerated roots that tested negative for CLas by real-time PCR, CLas was frequently detected from roots grown in nursery soil with autoclaving, after cultivation for a month or more. Conclusions: Latent infection with CLas was detectable by real-time PCR using root tissues regenerated by cuttings and roots grown in nursery soil with autoclaving. These results suggest that the new method of investigation would provide great opportunities for early detection of CLas in asymptomatic citrus trees from field surveys, and would accelerate the eradication practice of citrus greening.


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