Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling

Background The microbiota of the lower female genital tract plays an important role in women’s health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT. Methods DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition. Results DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.

Preparing Gene of interest for GateWay cloning (2 step PCR process) v2

GateWay recombination cloning is achieved by flanking your gene of interest with GateWay attachment sites. In our case attB1 and attB2. Those sites are added to the PCR product via primers with 5' extensions. Since those primes create 31 bp and 30 bp 5' primer extensions respectively, plus about 20 bp of actual binding primer sequence it becomes expensive fast if you need 2 x ~50 bp primers for every GOI. We therefore use a 2 step PCR process to attach GateWay attB1 and attB2 sites. We first run a gene specific PCR with primers carrying short 5' extesions, and then a second PCR utilizing universal GateWay primers which bind to the short extension of the first PCR product to create the full attB1 and attB2 sites. This protocol has been adapted from: 2-STEP GATEWAY PCR EXPERIMENTS

Sus sp. DNA ENCODING cyt b GENE DETECTION TEST ON SOFT GEL CANDY SAMPLES USING PCR METHOD

Softgel candy is soft-textured confectionery processed by the addition of several components such as gum, pectin, starch and gelatin, to obtain a supple product and packed after aging treatment first. Gelatin is one of the main components in the manufacture of soft candy derived from the hydrolysis of collagen connective tissue and animal bone that serves as the nature of gelling agents, stabilizers or emulsifiers. However, the gelatin used in products not yet labeled halal Indonesian Council of Ulama (MUI) is particularly vulnerable to pork gelatin, since pork gelatin is cheaper than cattle. The purpose of this study was to test the contaminants of pig DNA on 17 samples of soft candles not labeled halal MUI. This research used Polymerase Chain Reaction (PCR) method. Seventeen samples were isolated by DNA, then spectrophotometry was performed, followed by PCR. The PCR product is run electrophoresis. Visualize the DNA with a UV gel documentation. Primer used is primer gene encoding cyt b DNA pork. Results showed that 17 samples were negative contaminants, while the positive control of pork showed a DNA band of 149 bp. This shows that Softgel Candy 17 samples do not contain pork gelatin.

Frequencies of CYP2B6∗4,∗5, and ∗6 Alleles within an Iranian Population (Mazandaran)

Background. The human CYP2B subfamily consists of one functional gene (CYP2B6) and one pseudogene (CYP2B7P). Cytochrome P450 2B6 (CYP2B6) is a highly polymorphic enzyme that shows marked interindividual and interethnic variations. Currently, 38 alleles have been described, and some of the allelic variants have been associated with low enzyme activity. The aim of this study was to investigate the frequencies of CYP2B6∗4, CYP2B6∗5, and CYP2B6∗6 alleles in the Mazani ethnic group among Iranian Population. Methods. The study was conducted in 289 unrelated healthy volunteers. DNA was extracted from peripheral blood and analyzed by the PCR-RFLP protocol. The PCR product was digested with restriction enzymes and then separated using agarose gel electrophoresis. Results. The frequency of CYP2B6∗4, CYP2B6∗5, and CYP2B6∗6 in this study was 34.60%, 7.26%, and 34.54%, respectively. Conclusion. The frequency of the CYP2B6∗4 allele in the Mazani ethnic group was much higher (34.60%) than other population. The frequency of CYP2B6∗6 (34.54%) also was higher than its frequency in other previously reported population. But the frequency of CYP2B6∗5 in this study was lower than expected. These results will be useful in understanding the ethnic diversity in Iranian population and offer a preliminary basis for more rational use of drugs that are substrates for CYP2B6 in this population.

De Novo Assembly and Species-Specific Marker Development as a Useful Tool for the Identification of Scutellaria L. Species

Scutellaria L. (family Lamiaceae) includes approximately 470 species found in most parts of the world and is commonly known as skullcaps. Scutellaria L. is a medicinal herb used as a folk remedy in Korea and East Asia, but it is difficult to identify and classify various subspecies by morphological methods. Since Scutellaria L. has not been studied genetically, to expand the knowledge of species in the genus Scutellaria L., de novo whole-genome assembly was performed in Scutellaria indica var. tsusimensis (H. Hara) Ohwi using the Illumina sequencing platform. We aimed to develop a molecular method that could be used to classify S.indica var. tsusimensis (H. Hara) Ohwi, S. indica L. and three other Scutellaria L. species. The assembly results for S.indica var. tsusimensis (H. Hara) Ohwi revealed a genome size of 318,741,328 bp and a scaffold N50 of 78,430. The assembly contained 92.08% of the conserved BUSCO core gene set and was estimated to cover 94.65% of the genome. The obtained genes were compared with previously registered Scutellaria nucleotide sequences and similar regions using the NCBI BLAST service, and a total of 279 similar nucleotide sequences were detected. By selecting the 279 similar nucleotide sequences and nine chloroplast DNA barcode genes, primers were prepared so that the size of the PCR product was 100 to 1000 bp. As a result, a species-specific primer set capable of distinguishing five species of Scutellaria L. was developed.

Melting curve analysis to differentiate Rickettsia spp. and Rickettsia felis

Rickettsiosis, caused by Rickettsia species, is one of the old arthropod-borne illness that commonly found in humans and animals. One of the barriers to rickettsiosis control is the intricacy and time-consuming nature of rickettsiosis laboratory diagnosis. This study aimed to establish quantitative real-time PCR targeting the gltA gene for the DNA differentiation of Rickettsia spp. and Ricketsia felis. The collection of cat flea was extracted to acquire the DNA of Rickettsia. Primers were designed based on the analysis of Rickettsia gltA gene sequences. The confirmation of R. felis was performed by sequencing of PCR product. BLAST analysis was done to confirm the closest similarity of the sequences. Results of this study highlighted the melting temperature was reached at 78,5 °C for Rickettsia spp. and 76.5+0.5 °C for Rickettsia felis. The melting peak temperatures were significantly different between Rickettsia spp. and R. felis (p<0.05). The findings of this work are crucial in the development of powerful diagnostic procedures for detecting and distinguishing Rickettsia spp. and R. felis species.

Morphological and molecular differentiation between Culicoides oxystoma and Culicoides kingi (Diptera: Ceratopogonidae) in Tunisia

Background Culicoides kingi and Culicoides oxystoma belong to the Schultzei group of biting midges. These two species are vectors of disease in livestock of economic importance. As described in the literature, morphological identification for discrimination between them is still unclear. However, species-specific identification is necessary to solve taxonomic challenges between species and to understand their roles in disease transmission and epidemiology. This study aims to develop accurate tools to discriminate C. oxystoma from C. kingi using traditional morphometry and polymerase chain reaction-restriction fragment length polymorphism (PCR RFLP) assays for use in developing countries. Methods Specimens were collected from the region of Kairouan in central Tunisia. A total of 446 C. oxystoma/C. kingi individuals were identified using traditional morphometric analyses combined with PCR–RFLP of the cytochrome c oxidase subunit I gene. Thirteen morphometric measurements were performed from the head, wings, and abdomen of slide-mounted specimens, and six ratios were calculated between these measurements. Multivariate analyses of the morphometric measurements were explored to identify which variables could lead to accurate species identification. Results Four variables, namely antennae, wings, spermathecae, and palpus length, were suitable morphometric characteristics to differentiate between the species. Digestion with the SspI restriction enzyme of the PCR product led to good discriminative ability. Molecular procedures and phylogenetic analysis confirmed the efficiency of this simple and rapid PCR–RFLP method. Conclusions This study highlights for the first time in Tunisia the presence of C. oxystoma and its discrimination from C. kingi using abdominal measurements and the PCR–RFLP method. This approach could be applied in future epidemiological studies at the national and international levels. Graphical Abstract

sgRNA design for DLT gene editing using CRISPR-Cas9 and in-silico mutation prediction in Rice cv. Hawara Bunar

The DWARF AND LOW TILLERRING (DLT) gene is a transcription factor for a gene involved in Brassinosteroid (BR) biosynthesis. Manipulating BR biosynthesis will affect the height and tiller number of rice. CRISPR-Cas9 is an accurate tool to edit a gene sequence. The accuracy of site editing of the CRISPR-Cas9-mediated target gene editing is determined by the 20 nucleotide sequences in the sgRNA and the binding site known as the Protospacer Adjacent Motif (PAM). The study aimed to design sgRNA and predict the DLT gene mutation sites in rice cv. Hawara Bunar. The exon 1 of the DLT gene was amplified using a primer pair designed from the reference gene. The PCR product was then sequenced, and the sequence was used to design sgRNA. The study has designed sgRNA located on the targeted sequence that corresponds to the Gras family protein domain of the exon1 DLT gene. The mutation sites were predicted to be at the domain site through the alignment of the nucleotide and amino acid sequences of the DLT gene and the reference gene. It is predicted that mutations in the target site that corresponds to the protein domain will change the protein structure and its function.

Molecular Diagnosis of Fungal Sinusitis Using Limit of Detection

Background: As an inflammatory process that involves the paranasal sinuses, chronic sinusitis (CS) is one of the most prevalent chronic illnesses that affects all age groups. Parasitic fungi are involved in sinusitis infections. Objective: This study is aimed at the molecular detection of sinusitis caused by such fungi. Methods: Seventy-two samples were collected from the secretions of maxillary and frontal sinuses of patients from Rasoul-e Akram (PbUH) Hospital in Tehran during sinus operation. Fungal genomic DNA was extracted by a DNP kit. The detection of fungi was carried out by employing a sequence-specific target, namely mt cyte b gene locus, and using primers. Polymerase chain reaction (PCR) was optimized, and the limit of detection (LOD) and specificity tests were performed. The amplicon was cloned by the T/A cloning method, which was used for sequencing and positive control. Results: The 430-bp PCR product underwent appropriate propagation before being amplified and was observed on 1.5% electrophoreses gel. The evaluation of the selected primers with seven DNA constructs from another microorganisms demonstrated 100% specificity. The limit of detection of the optimized test was evaluated up to 50 fungi. Out of 72 samples, 9.7% were positive for fungi existence. Conclusions: This study indicated that molecular diagnosis of the target mt cyte b gene using LOD enhances clinical laboratory detection of fungal sinusitis.

Impact of a Novel W2027L Mutation and Non-Target Site Resistance on Acetyl-CoA Carboxylase-Inhibiting Herbicides in a French Lolium multiflorum Population

Herbicides that inhibit acetyl-CoA carboxylase (ACCase) are among the few remaining options for the post-emergence control of Lolium species in small grain cereal crops. Here, we determined the mechanism of resistance to ACCase herbicides in a Lolium multiflorum population (HGR) from France. A combined biological and molecular approach detected a novel W2027L ACCase mutation that affects aryloxyphenoxypropionate (FOP) but not cyclohexanedione (DIM) or phenylpyraxoline (DEN) subclasses of ACCase herbicides. Both the wild-type tryptophan and mutant leucine 2027-ACCase alleles could be positively detected in a single DNA-based-derived polymorphic amplified cleaved sequence (dPACS) assay that contained the targeted PCR product and a cocktail of two discriminating restriction enzymes. Additionally, we identified three well-characterised I1781L, I2041T, and D2078G ACCase target site resistance mutations as well as non-target site resistance in HGR. The non-target site component endowed high levels of resistance to FOP herbicides whilst partially impacting on the efficacy of pinoxaden and cycloxydim. This study adequately assessed the contribution of the W2027L mutation and non-target site mechanism in conferring resistance to ACCase herbicides in HGR. It also highlights the versatility and robustness of the dPACS method to simultaneously identify different resistance-causing alleles at a single ACCase codon.