Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to detect ultra low population ofRalstonia solanacearum(Smith 1896) Yabuchiet al.(1996)

Abstract

Aims: To develop a reliable and sensitive protocol for detection of Ralstonia solanacearum using MDA-PCR (Multiple displacement amplification-PCR amplification). Methods and Results: MDA-PCR technique was performed on pure cell lysates as well as soil samples. Pure cell lysate as well as that of soil DNA was used as template in MDA reaction. MDA of template DNA was carried out in the presence of sample buffer, reaction buffer and enzyme mix (U 29 DNA polymerase and random hexamers). The MDA amplified DNA was used for PCR amplification using R. solanacearum -specific PCR primers. MDA-PCR could detect as low as 1 colony forming unit (CFU ml )1 ) of bacteria within 8 h including DNA isolation. Conclusion: MDA followed by standard PCR facilitated the detection of pathogen from very low count samples. The method is of great importance in managing the brown rot disease of potato. Significance and Impact of study: The ultrasensitive detection technique developed in the present study is sensitive and speedy enough to be included into integrated wilt disease control programmes.

Apical Root Canal Microbiota as Determined by Reverse-capture Checkerboard Analysis of Cryogenically Ground Root Samples from Teeth with Apical Periodontitis

“…MDA has been shown to provide uniform amplification of the whole genome with extensive coverage, generate extremely long DNA products compared with previous whole genome amplification methods, and result in high DNA yields even starting from very low template amounts, with reduced amplification bias (27,28). It has been regarded as an important pre-PCR procedure for the detection of low copy number sequences (29,30). In addition, MDA provides enough amplified DNA to perform multiple analyses of the same sample.…”

A review of pest surveillance techniques for detecting quarantine pests in Europe

“…Grover et al . () used MDA‐PCR to detect ultra‐low populations; Smith and de Boer () developed an improved TaqMan method for PCR; Poussier et al . (), Alvarez Restrepo et al .…”

“…Laboratory detection methods are becoming increasingly sensitive. Grover et al (2009) used MDA-PCR to detect ultra-low populations; Smith and de Boer (2009) developed an improved TaqMan method for PCR; Poussier et al (2005), Alvarez Restrepo et al (2008) and Lin et al (2009) used a BIO-PCR protocol that can be selective and rapid (Paret et al, 2008;Kutin et al, 2009);Kubota et al (2008) developed a loop-mediated amplification of DNA (LAMP).…”

Rapid Method for Isolation of PCR Amplifiable Genomic DNA of <i>Ralstonia solanacearum</i> Infested in Potato Tubers

“…In order to have healthy seeds very sensitive and accurate detection methods are required. For quicker detection of the pathogen, immunodiagnostics [7] and polymerase chain reaction based tests [8,9] have been developed (based on PCR amplification of DNA and RNA). The PCR based techniques are more sensitive and specific as compared to immunodiagnostic methods and can be used for detection of ultra-low population of R. solanacearum in potato tubers [9].…”