Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR.
Colony PCR and semi-nested PCR techniques were employed for screening polyhydroxyalkanoate (PHA) producers isolated from the environment. Three degenerate primers were designed based on multiple sequence alignment results and were used as PCR primers to detect PHA synthase genes. Optimized colony PCR conditions were achieved by adding 3% DMSO combined with 1 M betaine to the reaction mixture. The sensitivity limit of the colony PCR was 1x 10(5) viable cells for Ralstonia eutropha. Nineteen PHA-positive bacteria were used to evaluate this PCR protocol; fifteen of the nineteen could be detected by colony PCR, and the other four could be detected by applying semi-nested PCR detection following colony PCR. In a preliminary screening project, 38 PHA-positive strains were isolated from environmental samples by applying the PCR protocol, and their phenotype was further confirmed by Nile blue A staining assay. By combining the colony PCR and semi-nested PCR techniques, a rapid, reliable and highly accurate detection method has been developed for detecting PHA producers. This protocol is suitable for screening large numbers of environmental isolates. The PHA accumulation ability of well-separated colonies isolated from environmental samples can be directly validated by PCR with no further culturing or chromosomal DNA extraction procedures. In addition to its application to the screening of wild-type isolates, the individual PCR-amplified product is also suitable as a specific probe for PHA operon cloning. The results suggest that the application of this PCR protocol for rapid detection of PHA producers from the environment is plausible.
Sheu
Der-Shyan
DS
Graduate Institute of Agricultural Chemistry, National Taiwan University, 1, Sec. 4, Roosevelt Rd, Taipei 106, Taiwan1.
Wang
Yun-Ting
YT
Graduate Institute of Agricultural Chemistry, National Taiwan University, 1, Sec. 4, Roosevelt Rd, Taipei 106, Taiwan1.
Lee
Chia-Yin
CY
Graduate Institute of Agricultural Chemistry, National Taiwan University, 1, Sec. 4, Roosevelt Rd, Taipei 106, Taiwan1.
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001482:Base Composition; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004783:Environmental Microbiology; D005069:Evaluation Studies as Topic; D005798:Genes, Bacterial; D006880:Hydroxy Acids / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D006880:Hydroxy Acids; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; C060970:poly-beta-hydroxybutyrate polymerase