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Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

ABSTRACT: An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine kidney tissue.

SUBMITTER: Magnusson HB

PROVIDER: S-EPMC202022 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

Magnússon H B HB Fridjónsson O H OH Andrésson O S OS Benediktsdóttir E E Gudmundsdóttir S S Andrésdóttir V V

Applied and environmental microbiology 19941201 12

An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine k ...[more]

PMID: 7529017

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Project description:BackgroundSequencing of the PCR-amplified 16S rRNA gene has become a common approach to microbial community investigations in the fields of human health and environmental sciences. This approach, however, is difficult when the amount of DNA is too low to be amplified by standard PCR. Nested PCR can be employed as it can amplify samples with DNA concentration several-fold lower than standard PCR. However, potential biases with nested PCRs that could affect measurement of community structure have received little attention.ResultsIn this study, we used 17 DNAs extracted from vaginal swabs and 12 DNAs extracted from stool samples to study the influence of nested PCR amplification of the 16S rRNA gene on the estimation of microbial community structure using Illumina MiSeq sequencing. Nested and standard PCR methods were compared on alpha- and beta-diversity metrics and relative abundances of bacterial genera. The effects of number of cycles in the first round of PCR (10 vs. 20) and microbial diversity (relatively low in vagina vs. high in stool) were also investigated. Vaginal swab samples showed no significant difference in alpha diversity or community structure between nested PCR and standard PCR (one round of 40 cycles). Stool samples showed significant differences in alpha diversity (except Shannon's index) and relative abundance of 13 genera between nested PCR with 20 cycles in the first round and standard PCR (P<0.01), but not between nested PCR with 10 cycles in the first round and standard PCR. Operational taxonomic units (OTUs) that had low relative abundance (sum of relative abundance <0.167) accounted for most of the distortion (>27% of total OTUs in stool).ConclusionsNested PCR introduced bias in estimated diversity and community structure. The bias was more significant for communities with relatively higher diversity and when more cycles were applied in the first round of PCR. We conclude that nested PCR could be used when standard PCR does not work. However, rare taxa detected by nested PCR should be validated by other technologies.

Project description:BackgroundAbout two out of three Ethiopians are at risk of malaria, a disease caused by the parasites Plasmodium falciparum and Plasmodium vivax. Anopheles stephensi, an invasive vector typically found in South Asia and the Middle East, was recently found to be distributed across eastern and central Ethiopia and is capable of transmitting both P. falciparum and P. vivax. The detection of this vector in the Horn of Africa (HOA) coupled with widespread insecticide resistance requires that new methods of vector control be investigated in order to control the spread of malaria. Wolbachia, a naturally occurring endosymbiotic bacterium of mosquitoes, has been identified as a potential vector control tool that can be explored for the control of malaria transmission. Wolbachia could be used to control the mosquito population through suppression or potentially decrease malaria transmission through population replacement. However, the presence of Wolbachia in wild An. stephensi in eastern Ethiopia is unknown. This study aimed to identify the presence and diversity of Wolbachia in An. stephensi across eastern Ethiopia.MethodsDNA was extracted from An. stephensi collected from eastern Ethiopia in 2018 and screened for Wolbachia using a 16S targeted PCR assay, as well as multilocus strain typing (MLST) PCR assays. Haplotype and phylogenetic analysis of the sequenced 16S amplicons were conducted to compare with Wolbachia from countries across Africa and Asia.ResultsTwenty out of the 184 mosquitoes screened were positive for Wolbachia, with multiple haplotypes detected. In addition, phylogenetic analysis revealed two superclades, representing Wolbachia supergroups A and B (bootstrap values of 81 and 72, respectively) with no significant grouping of geographic location or species. A subclade with a bootstrap value of 89 separates the Ethiopian haplotype 2 from other sequences in that superclade.ConclusionsThese findings provide the first evidence of natural Wolbachia populations in wild An. stephensi in the HOA. They also identify the need for further research to confirm the endosymbiotic relationship between Wolbachia and An. stephensi and to investigate its utility for malaria control in the HOA.

Dataset Information

Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

Publications

Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

Similar Datasets

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