ABSTRACT
IMPORTANCE: Plasmid size is one of the factors affecting transfection efficacy in most of the molecular genetic research studies. One effective approach for reducing plasmid size is to replace relatively large, conventional antibiotic resistance genes with the short-size dfrB10 gene. The successful construct of a series of dfrB10-based tool plasmids and their functional validation, via comparison with original plasmids, suggest that dfrB10 is a potent drug resistance selection marker. The antibiotic trimethoprim offers convenient usage comparable to that of ampicillin or kanamycin. Additionally, fluorescence analysis has demonstrated the compatibility of TMP with protein expression in various host cells. Based on these findings, TMP-dfrB10 could be an alternative choice for future use in molecular genetic research studies that require miniature plasmids to achieve optimal results.
Subject(s)
Anti-Bacterial Agents , Trimethoprim , Plasmids/genetics , Anti-Bacterial Agents/pharmacology , Drug Resistance, MicrobialABSTRACT
Dried blood spot (DBS) based PCR was considered an inexpensive and feasible method for detecting pathogens in the blood. The DBS carrier filter paper and PCR kits are crucial for accurate diagnosis. We evaluated 4 types of filter papers and 20 PCR kits for DBS samples. The PCR detecting Plasmodium results showed that the minimum detection limit of the 4 filter papers was 1 × 102 parasites/µL, and the positive rates of 20 PCR kits ranged from 0% to 100%. PCR results were satisfactory for detecting Plasmodium falciparum (P. falciparum) and Plasmodium. vivax (P. vivax) in archived DBS samples and Babesia gibsoni (B. gibsoni) in fresh pet DBS samples. Our results provided a useful reference for the detection of blood pathogens with DBS samples and direct PCR, especially for screening the cost-efficacy combination of filter paper and PCR kit in resource-limited areas.
