A Rapid Digital PCR System with a Pressurized Thermal Cycler

We designed a silicon-based fast-generated static droplets array (SDA) chip and developed a rapid digital polymerase chain reaction (dPCR) detection platform that is easy to load samples for fluorescence monitoring. By using the direct scraping method for sample loading, a droplet array of 2704 microwells with each volume of about 0.785 nL can be easily realized. It was determined that the sample loading time was less than 10 s with very simple and efficient characteristics. In this platform, a pressurized thermal cycling device was first used to solve the evaporation problem usually encountered for dPCR experiments, which is critical to ensuring the successful amplification of templates at the nanoliter scale. We used a gradient dilution of the hepatitis B virus (HBV) plasmid as the target DNA for a dPCR reaction to test the feasibility of the dPCR chip. Our experimental results demonstrated that the dPCR chip could be used to quantitatively detect DNA molecules. Furthermore, the platform can measure the fluorescence intensity in real-time. To test the accuracy of the digital PCR system, we chose three-channel silicon-based chips to operate real-time fluorescent PCR experiments on this platform.

A Model System for Sensitive Detection of Viable E. coli Bacteria Combining Direct Viability PCR and a Novel Microarray-Based Detection Approach

We established an innovative approach that included direct, viability, and nested PCR for rapid and reliable identification of the fecal indicator organism Escherichia coli (E. coli). Direct PCR enabled successful amplification of the target uidA gene, omitting a prior DNA isolation or purification step. Furthermore, we applied viability PCR (v-PCR) to ensure the detection of only relevant viable bacterial cells. The principle involves the binding of propidium monoazide (PMA), a selective nucleic acid intercalating dye, to accessible DNA of heat killed bacteria cells and, consequently, allows viable and heat killed E. coli cells to be discriminated. To ensure high sensitivity, direct v-PCR was followed by a nested PCR step. The resulting amplicons were analyzed by a rapid 30 min microarray-based DNA hybridization assay for species-specific DNA detection of E. coli. A positive signal was indicated by enzymatically generated silver nanoparticle deposits, which served as robust endpoint signals allowing an immediate visual readout. The presented novel protocol allows the detection of 1 × 101 viable E. coli cells per PCR run.

Applicability of human-specific STR systems, GlobalFiler™ PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex® Fusion 6C in chimpanzee (Pan troglodytes)

Objectives Human identification systems based on STRs are widely used in human population genetics and forensic analysis. This study aimed to validate the cross-reactivity of three widely known human-specific STR identification systems i.e. GlobalFiler™ PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex® Fusion 6C in chimpanzee. Results The present study revealed the successful amplification of 18 loci using GlobalFiler™ PCR Amplification Kit, 18 loci using Investigator 24plex QS Kit, and 20 loci using PowerPlex® Fusion 6C system. The marker Amelogenin (AMEL) showed differential allele size between male and female revealing the gender identity of chimpanzees and thus validates their application concerning forensic examination, population estimation, and genetic analysis.

Applicability of Human-specific STR systems, GlobalFiler™ PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex® Fusion 6C in Chimpanzee (Pan troglodytes)

Objectives The human identification systems based on STRs are widely used in human population genetics and forensic analysis. This study aimed to validate the cross-reactivity of three widely known Human-specific STR identification systems i.e. GlobalFiler™ PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex® Fusion 6C in Chimpanzee. Result The present study revealed the successful amplification of 18 loci using GlobalFiler™ PCR Amplification Kit, 18 loci using Investigator 24plex QS Kit, and 20 loci using PowerPlex® Fusion 6C system. The marker Amelogenin (AMEL) showed differential allele size between male and female revealing the gender identity of Chimpanzees and thus validates their application concerning forensic examination, population estimation, and genetic analysis.

Transferability of apple and pear SSRs to other temperate pome fruit crops of family Rosaceae

Extensive use of simple sequence repeat (SSR) is facilitated if loci would be transferable across species even in closely related genera to overcome high cost and efforts involved in their development as major constraints. In the present study, apple and pear genomic microsatellite primer pairs were used to amplify SSR loci in apple, pear, quince and loquat genotypes, respectively. Already reported SSRs were selected based on their polymorphic survey for successful amplification with at least one polymerase chain reaction (PCR) product of the approximate size expected for a homologous locus screened among apple and pear genotypes for further transferability exploration across other temperate pome fruit crops, respectively. Highest transferability of apple and pear SSR, 61.53 % and 73.33 % was observed in closely related quince and apple genotypes, respectively. This indicated that primer binding sites between these two closely related genera, Malus and Pyrus, are fairly well conserved. Maximum transferability rate was found to be 93.33 % and 80.00 % across all the subjected genotypes for primer CH05D11 and TSUenh016 in apple and pear, respectively. The transferability of markers is based on genomic similarity, and can reflect the relationship of genome collinearity and even evolution between species. This high level of transferability of apple and pear SSRs to other temperate pome fruit crops indicated their promise for application to future molecular screening, map construction, and comparative genomic studies, etc.

Fumonisin B1 Production by Fusarium Species and Mycotoxigenic Effect on Larval Zebrafish

Fumonisin B1 (FB1) is a common mycotoxin produced by Fusarium species particularly F. proliferatum and F. verticillioides. The toxin produced can cause adverse effects on humans and animals. The objectives of this study were to detect the production of FB1 based on the amplification of FUM1 gene, to quantify FB1 produced by the isolates using Ultra-fast Liquid Chromatography (UFLC) analysis, to examine the embryotoxicity effect of FB1 and to determine EC50 toward the larvae of zebrafish (Danio rerio). Fifty isolates of Fusarium species were isolated from different hosts throughout Malaysia. Successful amplification of the FUM1 gene showed the presence of this gene (800 bp) in the genome of 48 out of 50 isolates. The highest level of FB1 produced by F. proliferatum isolate B2433 was 6677.32 ppm meanwhile F. verticillioides isolate J1363 was 954.01 ppm. From the assessment of embryotoxicity test of FB1 on larvae of zebrafish, five concentrations of FB1 (0.43 ppm, 0.58 ppm, 0.72 ppm, 0.87 ppm and 1.00 ppm) were tested. Morphological changes of the FB1 exposed-larvae were observed at 24 to 168 hpf. The mortality rate and abnormality of zebrafish larvae were significantly increased at 144 hpf exposure. Meanwhile, the spontaneous tail coiling showed a significant difference. There were no significant differences in the heartbeat rate. As a conclusion, the presence of FUM1 in every isolate can be detected by FUM1 gene analysis and both of the species produced different concentrations of FB1. This is the first report of FB1 produced by Fusarium species gave a significant effect on zebrafish development.

Sequencing of Dna Isolated From Colorectal Sample Fixed in Liquid Formalin : Obstacles and Required Modifications

The necessary modifications in the protocol of general purpose DNA isolation kit to isolate and amplify a target region of genome from colorectal cancer tissues fixed in liquid formalin were made. It is shown that a one hour digestion with proteinase K yields enough DNA from formalin fixed colorectal tissue for subsequent PCR and sequencing. Moreover, using 100% ethanol instead of standard 50% during DNA binding step in the column improves the yield. As DNA fragmentation is unavoidable in formalin fixed tissue, PCR protocol was modified by increasing polymerase concentration to get successful amplification. Following these modifications, two regions of KRAS and BRAF genes were amplified and successfully sequenced from three different patients. These modifications provide a low cost option for Sanger sequencing of DNA isolated from formalin fixed tissue. Dhaka Univ. J. Biol. Sci. 29(2): 165-174, 2020 (July)

Development and characterization of thirteen novel microsatellite markers for use in Greenland sharks (Somniosus microcephalus), with cross-amplification in Pacific sleeper sharks (Somniosus pacificus)

Objective The objectives of this research are to isolate, develop and characterize polymorphic microsatellite markers for use in Greenland sharks ( Somniosus microcephalus ). Despite utility in population analyses, microsatellite markers have not been previously developed for this species. Development of these markers, and successful amplification in closely related Pacific sleeper sharks ( S. pacificus ), will facilitate research in the genetic variation of contemporary and future populations of sleeper shark species. Results Thirteen microsatellite loci were successfully amplified and yielded multi-locus genotypes for 32 S. microcephalus individuals from Grise Fjord (n = 16) and Svalbard (n = 20). Each locus yielded between 2 to 9 alleles and observed heterozygosity ranged from 0.11 to 0.70 when estimated across both sites. One locus and three loci deviated from HWE following Bonferroni correction, for individuals sampled from Grise Fjord and Svalbard, respectively. Cross-amplification was successful at every locus for five of the ten S. pacificus individuals.

Barcoding of ToLCV Resistant Tomato Germplasm in Bangladesh

The current study was carried out to confirm the existence of the ToLCV resistant genes (Ty-1 to Ty-5) in the germplasm using molecular markers and to identify at the genomic level following phylogenetic relationships analysis among some local tomato germplasm using DNA barcoding. Most of the tomato germplasm (Ten out of 14) contain the dominant Ty-genes as revealed by PCR analysis. “Barcoding” of the non-coding plastid trnH-psbA intergenic spacer region, three plastidal regions: rbcL, rpoB, rpoC1, spacer region of nuclear genome ITS and a mitochondrial region matK were employed following PCR and sequence analysis of the germplasm. Among all the barcode genes, rpoB, rbcL, trnH-psbA and ITS were leading candidates for successful amplification and used for the identification of the germplasm as S. lycopersicum in multi-locus identification based on their sequences. Neighbor-Joining phylogenetic tree was constructed in which the germplasm were clustered into five main clades. The current study was successful to establish an efficient barcoding protocol for the correct identification of tomatoes and was capable of establishing elite gene source(s) for biotic stress resistance tomato varieties which would serve as potential donor plants in modern breeding programs. Plant Tissue Cult. & Biotech. 30(1): 107-117, 2020 (June)

Identification of Torque Teno Virus/Torque Teno-Like Minivirus in the Cervical Lymph Nodes of Kikuchi-Fujimoto Lymphadenitis Patients (Histiocytic Necrotizing Lymphadenitis): A Possible Key to Idiopathic Disease

Kikuchi-Fujimoto disease (KFD) is rare, and many infectious agents have been suspected for its etiology. This report presents an interesting case of KFD found with torque teno virus/torque teno minivirus (TTV/TTMV), which closely resembles the circovirus that causes necrotizing lymphadenitis in pigs. Three Korean patients showed several enlarged lymph nodes in their neck. Quantitative polymerase chain reaction (qPCR) and subsequent DNA sequencing for TTV/TTMV using formalin-fixed paraffin-embedded tissue were performed. Histologic examination demonstrated typical features of KFD. qPCR showed successful amplification of TTV/TTMV, and DNA sequencing confirmed the results. It is the first report of TTV/TTMV presence in three patients with KFD.