Rapid and Specific Detection of Burkholderia glumae in Rice Seed by Real-Time Bio-PCR Using Species-Specific Primers Based on an rhs Family Gene

Abstract

In this study, we developed a reliable, quick, and accurate quantitative polymerase chain reaction (qPCR) assay to detect grain rot caused by Burkholderia glumae in rice seed. The control of bacterial grain rot is difficult, and the only practical methods for disease management rely on the use of pathogen-free seed, appropriate culture practices, and resistant cultivars. Therefore, the specific detection of this pathogen in seed is essential for effective control of the disease. However, other Burkholderia spp. are also detected by currently available molecular and serological methods. In this study, we exploited the available genome sequence information in public databases to develop specific PCR primers for accurate diagnosis of B. glumae. An SYBR Green real-time PCR primer set was designed based on the rhs family gene (YD repeat protein) of B. glumae BGR1 because these genes are structurally diverse. The specificity of the primers was evaluated using purified DNA from 5 isolates of B. glumae, 6 different species of Burkholderia, and 18 other reference pathogenic bacteria. The assay was able to detect at least one genome equivalent of cloned amplified target DNA using purified DNA or 1 CFU per reaction when using calibrated cell suspension. This method is rapid and reliable and has great potential for analyzing large numbers of samples.

Molecular identification of lactobacilos of the digestive tract of piglet (Sus scrofa domesticus)

“…En busca de bacterias ácido lácticas del tracto digestivo del lechón recién destetado, nos basándonos en el benéfico de los microorganismos naturales benéficos en el tracto digestivo del lechón (Álvarez, 2004;Figuero y Sánchez, 2016;Bertullo, 2001;Lessi, 1994;García et al, 2012;Partanen y Mroz, 1999;Buhnik-Rosenblau et al, 2012) La microbiota nativa tiene efectos benéficos (Guarner et al, 2008;Zhang et al, 2011;Borruel 2007), Podemos dar cuenta de organismos beneficiosos que encontramos en el tracto digestivo del lechón según Metzler y Mosenthin, 2009;Kim et al, 2012;Konstantinov et al, 2008;Pieper et al, 2006. De los microorganismos naturales con capacidad benéficas que se encuentran como bacterias cultivables naturales del tracto digestivo del lechón fueron diferenciadas 67 cultivables, inicialmente identificadas como bacterias acido lácticas semejante a los reportados por Jurado et al, 2013;Rosmini et al, 2004;Sánchez et al, 2015;Cranwell, 1985;Álvarez, 2004;Sánchez y Ochoa, 2016;Bertullo, 2001;Lessi, 1994;García et al, 2012;Partanen y Mroz, 1999;Buhnik-Rosenblau et al, 2012, tanto el Lactobacillus fermentus y Lactococcus lactis pertenecen al orden Lactobacillales a l igual a los encontrados para mejorar la producción de cerdos como son los Bacillus subtilis, Clostriduim butyricum, Lactobacillus rhamnosus, B. licheniformis y B. subtilis (Bavera et al, 2002;Gatti, 2013;Carro y Ranillas, 2002;Agudelo, 2014;Pérez, 2007;Guerra, 2005;Metzler y Mosenthin, 2009;…”

“…La retirada de los antibióticos como promotores de crecimiento en la UE desde enero de 2006, ha creado la necesidad de avanzar en la búsqueda de otros aditivos nutricionales no antibióticos (Metzler, 2010). Debido a esto, se ha estudiado la influencia de diversos microorganismos, en especial de bacteria nativas del género Lactobacillus, sobre la eficiencia productiva del lechón (Cueto-Vigil et al, 2010;Kim et al, 2012) A fin de solucionar la problemática formulada, se planteó la hipótesis de encontrar Lactobacilos en el tracto digestivo del lechón Sus escrofa domesticus.…”

Detection and Control Bacteria Cause Grain Rot Burkholderia glumae on Rice

“…Besides being able to detect the presence of B. glumae, SMART is also able to recognize the ecology and epidemiology of the target bacteria. Kim et al (2012) stated that the detection procedure for B. glumae bacteria could be done through the bio-PCR method. Previously, Sayler et al (2006) have developed an effective PCR method for detecting B. glumae in seeds and in plants.…”

Comparative analysis of microbial diversity and bacterial seedling disease‐suppressive activity in organic‐farmed and standardized commercial conventional soils for rice nursery cultivation

“…Real-time PCR was performed according to the procedure published by Kim et al (2012) using an ABI 7500 Fast Real-Time PCR System (ABI, Foster City, CA, USA). Relative amounts of PCR products were calculated according to the 2 −Δ(ΔCt) method (Livak, Thomas, & Schmittgen, 2001), and the products were also detected by 2% agarose gel electrophoresis according to the standard protocol (Sambrook & Russell, 2001).…”