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Rapid and Accurate Species-Specific PCR for the Identification of Lethal Chironex Box Jellyfish in Thailand.

ABSTRACT: Box jellyfish are extremely potent venom-producing marine organisms. While they have been found worldwide, the highest health burden has been anticipated to be the tropical Indo-Pacific of Southeast Asia (SEA). At least 12 Cubozoan species have now been documented in Thai waters, and many of them inflict acutely lethal strings, especially those under the order Chirodropida. Our previous study has successfully differentiated species of box jellyfish using DNA sequencing to support the morphological study. In this study, we specifically designed polymerase chain reaction (PCR) primers for the 16S ribosomal RNA (rRNA) gene and the mitochondrial DNA cytochrome oxidase subunit I (COI) gene of lethal Thai Chironex species. The SYBR green-based real-time PCR panel was performed for rapid species identification. The sensitivity and specificity of the panel were determined by testing samples of different species. Moreover, we applied the panel to the tentacle sample from a real patient, which helped confirm the animal-of-cause of envenomation. Our results show a success for species identification of box jellyfish using 16S rRNA and COI PCR panel, which revealed congruence between molecular and morphological identification. Furthermore, the panel worked very well with the unknown samples and jellyfish tissue from the real envenomation case. The results demonstrated that molecular panels were able to identify three species of Chironex box jellyfish both rapidly and accurately, and can be performed without having a complete specimen or morphological study.

SUBMITTER: Sathirapongsasuti N

PROVIDER: S-EPMC7796029 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score genotypes. ASQ is cost-effective because universal fluorescent probes negate the necessity of designing expensive probes for each locus.

Dataset Information

Rapid and Accurate Species-Specific PCR for the Identification of Lethal Chironex Box Jellyfish in Thailand.

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