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A multi-target, non-infectious and clonable artificial positive control for routine PCR-based assays.

ABSTRACT: Positive controls are essential for PCR reliability and are challenging to obtain for rare, exotic and/or emerging pathogens and pose biosafety risks if manufactured using infectious pathogens. Custom synthetic DNA inserts can be designed de novo in tandems of forward and reverse complement priming sequences to be inserted in circular plasmid vectors. To test this concept, artificial positive controls (APCs) for use in PCR were synthesized to contain primer sequences targeting four viruses (Barley yellow dwarf virus, Soilborne wheat mosaic virus, Triticum mosaic virus and Wheat streak mosaic virus) pathogenic to wheat and, as internal control, the plant mitochondrial nad5 gene. Thermodynamics and folding parameters of twenty-four APC inserts were assessed in silico. Two thermodynamically different APCs, designated optimal and sub-optimal, were cloned and tested using end point PCR. The optimal APC had a 100% amplification rate, while only 92% of virus-infected plant tissues, commonly used as reference positive controls, amplified. An array of APC priming sequences from different organisms and/or previously tested primers can be accommodated in a large and flexible number of positive control targets. APCs will streamline and standardize routine PCR, improve reliability and biosafety, and create opportunities for development and commercialization of new synthetic positive control sequences.

SUBMITTER: Caasi DR

PROVIDER: S-EPMC7114305 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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A multi-target, non-infectious and clonable artificial positive control for routine PCR-based assays.

Caasi Donna Ria J DR Arif Mohammad M Payton Mark M Melcher Ulrich U Winder Louise L Ochoa-Corona Francisco M FM

Journal of microbiological methods 20130905 2

Positive controls are essential for PCR reliability and are challenging to obtain for rare, exotic and/or emerging pathogens and pose biosafety risks if manufactured using infectious pathogens. Custom synthetic DNA inserts can be designed de novo in tandems of forward and reverse complement priming sequences to be inserted in circular plasmid vectors. To test this concept, artificial positive controls (APCs) for use in PCR were synthesized to contain primer sequences targeting four viruses (Barl ...[more]

PMID: 24013035

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Project description:The identification of sewage contamination in water has primarily relied on the detection of human-associated Bacteroides using markers within the V2 region of the 16S rRNA gene. Despite the establishment of multiple assays that target the HF183 cluster (i.e., Bacteroides dorei) and other Bacteroides organisms (e.g., Bacteroides thetaiota omicron), the potential for more human-associated markers in this genus has not been explored in depth. We examined the Bacteroides population structure in sewage and animal hosts across the V4V5 and V6 hypervariable regions. Using near-full-length cloned sequences, we identified the sequences in the V4V5 and V6 hypervariable regions that are linked to the HF183 marker in the V2 region and found these sequences were present in multiple animals. In addition, the V4V5 and V6 regions contained human fecal marker sequences for organisms that were independent of the HF183 cluster. The most abundant Bacteroides in untreated sewage was not human associated but pipe derived. Two TaqMan quantitative PCR (qPCR) assays targeting the V4V5 and V6 regions of this organism were developed. Validation studies using fecal samples from seven animal hosts (n = 76) and uncontaminated water samples (n = 30) demonstrated the high specificity of the assays for sewage. Freshwater Bacteroides were also identified in uncontaminated water samples, demonstrating that measures of total Bacteroides do not reflect fecal pollution. A comparison of two previously described human Bacteroides assays (HB and HF183/BacR287) in municipal wastewater influent and sewage-contaminated urban water samples revealed identical results, illustrating the assays target the same organism. The detection of sewage-derived Bacteroides provided an independent measure of sewage-impacted waters.IMPORTANCE Bacteroides are major members of the gut microbiota, and host-specific organisms within this genus have been used extensively to gain information on pollution sources. This study provides a broad view of the population structure of Bacteroides within sewage to contextualize the well-studied HF183 marker for a human-associated Bacteroides The study also delineates host-specific sequence patterns across multiple hypervariable regions of the 16S rRNA gene to improve our ability to use sequence data to assess water quality. Here, we demonstrate that regions downstream of the HF183 marker are nonspecific but other potential human-associated markers are present. Furthermore, we show the most abundant Bacteroides in sewage is free living, rather than host associated, and specifically found in sewage. Quantitative PCR assays that target organisms specific to sewer pipes offer measures that are independent of the human microbiome for identifying sewage pollution in water.

Dataset Information

A multi-target, non-infectious and clonable artificial positive control for routine PCR-based assays.

Publications

A multi-target, non-infectious and clonable artificial positive control for routine PCR-based assays.

Similar Datasets

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