A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens

Differentiation between Bursaphelenchus xylophilus and other related, non-pathogenic species can be ambiguous when based exclusively on morphological characters. The morphology of B. mucronatus and B. fraudulentus most closely resembles that of B. xylophilus. Moreover, all of these nematodes are found in both Asia and Europe and can colonise various species of pine. Therefore, for phytosanitary purposes it is necessary to identify the three species precisely and rapidly. We report the results of a multiplex PCR that utilises four primers to identify and discriminate the three Bursaphelenchus species simultaneously. The multiplex PCR yielded DNA fragments of 767, 305 and 132 bp, for B. xylophilus, B. mucronatus and B. fraudulentus, respectively. This primer combination has produced reliable results in multiplex PCR assays with a number of different populations of the listed species, and no cross-reactions were observed with other Bursaphelenchus species. The described approach is simple, reliable and cheaper than other molecular methods presently used for simultaneous identification of the above three species within the xylophilus group.

Download Full-text

One-Step Multiplex PCR for Simultaneous Detection and Identification of Eight Medically Important <i>Candida</i> Species

Open Journal of Stomatology ◽

10.4236/ojst.2021.111002 ◽

2021 ◽

Vol 11 (01) ◽

pp. 14-24

Author(s):

Akira Fukatsu ◽

Osamu Tsuzukibashi ◽

Hidenori Suzuk ◽

Katsuhiro Asaka ◽

Yoshinori Ono ◽

...

Keyword(s):

Multiplex Pcr ◽

Simultaneous Detection ◽

Detection And Identification ◽

One Step

Download Full-text

Multiplex PCR for detection of water-borne bacteria

Water Science & Technology Water Supply ◽

10.2166/ws.2016.126 ◽

2016 ◽

Vol 17 (1) ◽

pp. 169-175

Author(s):

Roohollah Kheiri ◽

Reza Ranjbar ◽

Mojtaba Memariani ◽

Leili Akhtari

Keyword(s):

Multiplex Pcr ◽

Target Genes ◽

Simultaneous Detection ◽

Diagnostic Procedures ◽

Molecular Techniques ◽

Detection Sensitivity ◽

Detection Methods ◽

Salmonella Spp ◽

Pcr Assays ◽

Water Borne

Microbial water-borne diseases still affect developing countries and are major water quality concerns throughout the world. Routine culture-based methods of identifying bacterial pathogens in water sources are laborious and time-consuming. Recently, the use of molecular techniques such as the polymerase chain reaction (PCR) has provided rapid and highly promising detection methods. In this study, we developed two multiplex PCR assays for simultaneous detection of six water-borne bacteria. Two triplex PCR protocols were developed to detect six target genes. The first protocol targets uidA (Escherichia coli), int (Shigella spp.), and gyrB (Pseudomonas aeruginosa) genes, while invA (Salmonella spp.), ompW (Vibrio cholera), and lacZ (coliforms) were amplified by the second protocol. Specificity testing was carried out for 12 reference strains. Furthermore, the applicability of the multiplex PCR assays for detection of these bacteria was investigated for 52 surface water samples. The results indicated that all primer pairs showed specificities only for their corresponding target organisms. The detection sensitivity of both multiplex PCR assays was 3 × 102 − 3 × 103 colony forming units. The developed assays represent simple and efficient diagnostic procedures for co-detection of water-borne bacteria and have the potential to provide earlier warnings of possible public health threats and more accurate surveillance of these organisms.

Download Full-text