The common methods that are presently used to identify Vibrio harveyi include microscopic examination and biochemical, immunological and PCR-based assays. These methods require technical expertise, and can be time-consuming. A rapid method is required for the high-throughput screening of large number of samples. As such, we have developed a rapid, simple yet sensitive and specific detection method based on the use of the loop-mediated isothermal amplification (LAMP) of DNA. A set of six primers, i.e., two outer, two inner and two loop primers, was designed based on the in silico analysis of a large pool of 39 strains of the toxR gene sequence of V. harveyi. The addition of the loop primers decreased the reaction time of the LAMP by more than half. Furthermore, with the application of SYBR Green, the result can be obtained as quickly as in 10 to 15 min without the need of gel electrophoresis. The specificity of the method primers was then determined by performing LAMP with Vibrio and non-Vibrio samples. LAMP has a greater sensitivity than PCR reaction. The sensitivity of PCR was at 0.6 pg concentration of V. harveyi recombinant plasmid DNA standard, while LAMP was able to detect lower amounts even at 0.6 fg. The development of the LAMP assay will provide a valuable tool for the high-throughput rapid detection of V. harveyi contamination both in laboratories and in the field.
Harumanis is one of the main signatures of Perlis with regards to its delightful taste, pleasant aroma and expensive price. Harumanis authenticity and productivity had become the remarks among the farmers, entrepreneurs, consumers and plant breeders due to the existence of morphological characteristics variation among the fruits and high production cost. Assessment of Harumanis morphological characteristics of natural population and different tree ages may represent a possible source of important characteristics for development and breeding purposes of Harumanis. The aim of this study is to evaluate the morphological variation of Harumanis collected from different location in Perlis and tree age. A total of 150 Harumanis fruits from 50 trees with three different stages of development (young, middle-aged and old) were characterised using 11 traits; 10 quantitative and one qualitative morphological trait. The ANOVA analyses in combination with Dunn's pairwise and Kruskal-Wallis multiple comparison test able to point out the existence of environmental factor and age influence towards the significant different of identified morphological traits except for Total Soluble Solid (TSS) and pulp percentage. Five clusters of 50 Harumanis accessions reflect a grouping pattern which not according to neither geographical region nor age. The result of Principal Component Analysis (PCA) using the first two principal components (PCs) provided a good approximation of the data explaining 84.09% of the total variance which majorly contributed by parameters of weight, fruit dimensional characteristics, peel percentage and hue angle, h. Preliminary screening of important morphological characteristics which contribute to the phenotypic diversity of Harumanis is successfully achieved. The findings can be employed by the plant breeders and farmers for the establishment of standard grading of Harumanis and advancement of breeding crop of Harumanis in future.