35 research outputs found
A novel loop-mediated isothermal amplification approach for sex identification of Columbidae birds
Because it is difficult to differentiate male and female Columbidae birds (e.g., Columba livia) on the basis of morphology,
detection of DNA fragments associated with Chromobox-Helicase-DNA binding genes or female-specific genes have been widely
used. The objective was to establish a loop-mediated isothermal amplification system involving the 18S ribosomal RNA gene and
a female-specific gene for sex identification of Columba livia birds. Unlike polymerase chain reaction (PCR), random amplification
polymorphic DNA-PCR and amplified fragment length polymorphism-PCR, target DNA was amplified under isothermal
conditions (the entire process was completed in 60 min). By modulating various parameters involved in amplification, e.g.,
concentrations of MgSO4, betaine, Bst polymerase, and deoxynucleotide triphosphates, as well as the relative ratio of outer/inner
primers and temperatures, optimal conditions for both targets were established that had equal detection limits (62.5 ng). To
simplify sex determination, direct observations of the presence of white precipitate (derived from magnesium pyrophosphates)
were used for positive samples, which was compared with the whitish ring which formed in a negative sample after addition of
CuSO4. This approach was a rapid alternative to electrophoresis or turbidimetry. DNA extracted from the blood and feathers of
various birds were tested using loop-mediated isothermal amplification; results were consistent with a standard PCR. Thus, the
assay was a simple, accurate, fast, and economical alternative suitable for veterinary practice