Efficient Method for Molecular Characterization of the 5′ and 3′ Ends of the Dengue Virus Genome

Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 5′ and 3′ ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 5′ and 3′ ends of dengue virus types 1 to 4. The 5′ and 3′ ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 5′ end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 3′ end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 5′ and 3′ ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods.

Comparison of Two Multiplex PCR Systems for Meat Species Authentication

Background: Meat species adulteration has become a problem of concern. This study aimed to compare two previously published multiplex Polymerase Chain Reaction (PCR) methods for meat species authentication. Methods: The primers used in the first multiplex PCR involved species-specific reverse primer for sheep, goat, cattle, pig, and donkey with universal forward primer. In the second multiplex PCR, the primers included species-specific forward and reverse primer for pork, lamb, ostrich, horse, and cow. The extracted DNA was then amplified with species-specific primers and with mix primers separately in the respective multiplex PCR. Results: The first multiplex PCR was accompanied with cross reactivity, whereas the second multiplex PCR was specific as expected for pork, lamb, ostrich, horse, and cow. The first set of multiplex PCR showed not always amplification of all species-specific DNAs with a mixture of DNA from mentioned animals. Regarding the second set of primers, the extracted DNA of different meat species was amplified with corresponding species primers as simplex PCR resulting in specific amplicons for species DNA prepared from sheep, ostrich, horse, pig, and cattle with the specific PCR products of 119, 155, 253, 100, and 311 bp, respectively. Conclusion: Based on the present investigation, we recommend the multiplex PCR with the second set of primers included species-specific forward and reverse primers for species authentication of five meat types, including pork, lamb, ostrich, horse, as well as cow.

Development of Tools for T Cell Repertoire Analysis (TCRB Spectratyping) for the Canine Model of Hematopoietic Cell Transplantation.

Analysis of the recombinatorial diversity of rearranged T cell receptor genes in mature T cells is an essential tool in the evaluation of immune status and immune reconstitution in hematopoietic cell transplantation studies. While the spectratyping technique is available in human clinical research and mouse models, the canine genome has not been sufficiently annotated to simply implement the assay on the dog TCRB locus. To this end, we have annotated by bioinformatics and experimentally all canine TCRBV segments, as well as TCRBD, TCRBC and most of TCRBJ segments. Of all 31 canine TCRBV found, 23 were functional and 8 were pseudogenes. A multiplex PCR-based assay was further designed to analyze the entire TCRBV spectratype in a set of 4 reactions each containing 4 to 5 V-segment specific forward primers and a common C-segment specific reverse primer. Direct sequencing of RT-PCR products confirmed that all amplified genes originated from predicted V-segments and that the designed V-specific PCR primers did not cross-react with other TCRBV family members. The usefulness of the spectratyping technique for canine model of transplantation was further demonstrated in analysis of T-cell repertoire reconstitution of irradiated dogs at different time points of recovery. Moreover, our simple and rapid V (J) annotation strategy relies on internet resources open to the general public and does not require specialized training in bioinformatics. It can be readily applied for de novo identification and mapping of TCRB gene families in other animal species where genome sequence drafts become available. Figure 1. TCRB spectratype of fetal canine thymus. (A) RT-PCR using TCRBV family-specific forward primers (V1 through V26) and a common C region-specific reverse primer. (B) Amplification products specific for family V1 (high abundance) and V26 (low abundance) were copied in run-off reactions with the fluorescent C-specific primer and resolved on capillary gel. Figure 1. TCRB spectratype of fetal canine thymus. (A) RT-PCR using TCRBV family-specific forward primers (V1 through V26) and a common C region-specific reverse primer. (B) Amplification products specific for family V1 (high abundance) and V26 (low abundance) were copied in run-off reactions with the fluorescent C-specific primer and resolved on capillary gel.