Development of a species-specific PCR-RFLP targeting rpoD gene fragment for discrimination of Aeromonas species

Abstract

This method provides an alternative method for laboratory identification, surveillance and epidemiological investigations of clinical and environmental specimens.

Methods for the diagnosis of bacterial fish diseases

“…For example, Hiney et al (1992) reported a polymerase chain reaction (PCR) that was capable of detecting ~ 2 cells of A. salmonicida, whereas Høie et al (1997) detected 10 3 and 10 4 colony forming units (CFUs) of A. salmonicida in 100 ml of kidney suspension with 16S rRNA sequencing and plasmid primers, respectively. Subsequent developments included nested PCR (Taylor and Winton 2002), terminal-restriction fragment length polymorphism (RFLP) (Nilsson and Strom 2002), PCR-RFLP (Puah et al 2018), multiplex PCR (Chapela et al 2018), real-time PCR (Keeling et al 2013), quantitative real-time-PCR (Du et al 2017), real-time recombinase polymerase amplification (Pang et al 2019) and reverse transcription-multiplex PCR (Rattanachaikunsopon and Phumkhachorn 2012). All these techniques reported extremely high levels of sensitivity, detecting numbers of cells well below the level associated with occurrences of clinical disease (Austin and Austin 2016).…”

Isolation, molecular characterization and antimicrobial susceptibility ofAeromonasspp. obtained from Tiger Grouper (Epinephelus fuscoguttatus) and Marble Goby (Oxyeleotris marmoratus) fish in Sabah, Malaysia

“…Using the amplified rpoD gene sequence and digested with Alu I, the species of individual isolates was determined (Puah, Khor, et al, 2018). The digested products were analysed using Agilent automated electrophoresis system and Agilent 2,100 Bioanalyzer (Agilent Technologies) and patterns generated (Figure 2) were analysed by Agilent 2,100 Expert Software.…”

“…The digested products were analysed using Agilent automated electrophoresis system and Agilent 2,100 Bioanalyzer (Agilent Technologies) and patterns generated (Figure 2) were analysed by Agilent 2,100 Expert Software. Species of individual isolates was determined by comparing RFLP patterns with the fragment sizes of the reference strains of all Aeromonas species (Puah, Khor, et al, 2018).…”

“…Taxonomy of the genus Aeromonas is complex, expansive and includes approximately 36 species to date (Puah, Khor, et al, 2018). In the past, this genus was misidentified as belonging to Vibrio or Plesiomonas in the family Vibrionaceae but recently it has been reclassified under Aeromonadaceae (Janda & Abbott, 2010).…”

“…Species identification can be achieved via sequencing investigation on rpoD housekeeping gene, a well‐documented molecular marker that has better resolving power than 16S rRNA (Puthucheary, Puah, & Chua, 2012). In 2018, Puah et al demonstrated that using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) targeting rpoD gene and single restriction endonuclease Alu I had enabled the differentiation of 25 Aeromonas species, offering an alternative method for species identification (Puah, Khor, et al, 2018).…”