Amplification Success Rate Research Articles

Comprehensive Evaluation of Cryptic Juglans Genotypes: Insight from Molecular Markers and Phylogenetic Analysis

Background/Objectives: The current research work aimed to evaluate the cryptic walnut genotypes of the Hazara region in Pakistan by using DNA barcoding and phylogenetic analysis. Methods: Based on morphological traits such as nut size, nut shape, and the number of leaflets, five genotypes were chosen and samples were collected for the current study. For molecular analysis, gDNA was isolated from the fresh leaves, and the five most effective angiosperm-specific markers, ITS2, rbcLa, rbcLc, rpoC1, and UBE3, were utilized. Based on amplification, sequencing, and identification success rates, ITS2 and UBE3 were recorded as the most efficient markers followed by rbcLa, rbcLc, and rpoC1. Results: During phylogenetic analysis, the query genotype-1 based on ITS2 and genotype-2 based on UBE3 clustered with (KF454101.1-Juglans regia) and (KC870919.1-J. regia) with bootstraps of 56 and 100, respectively. Genotype-3 based on rbcla clustered in a major clade with J. regia L., cultivars (MN397935.1 J. regia ‘Vina’) and (MN397934.1-J. regia ‘Serr’), (MN397933.1 J. regia ‘Pedro’), (MN397932.1 J. regia ‘Lara’), (MN397931.1 J. regia ‘Howard’), and (MN397930.1 J. regia ‘Hartley’) with bootstrap of 100. Meanwhile, genotype-4 and genotype-5 based on rbclc and rpoC1 clustered with (MN397935.1 J. regia ‘Vina’) and (MN397934.1 J. regia ‘Serr’), across the database sequences. To clarify the taxonomic status of cryptic walnut genotypes, it is necessary to combine diverse DNA barcodes. The results of ITS2 and UBE3, followed by rbcL barcoding markers, are promising taxonomic tools for cryptic walnut genotypes in Pakistan. Conclusions: It has been determined that the genotypes of walnuts in the study area are both J. regia L. and its cultivars and that the accuracy of discrimination regarding the genus Juglans L. is greater than 90%. The reported DNA barcodes are recommended for the correct identification and genetic evaluation of Juglans taxa and its population.

Technically feasible solutions to challenges in preimplantation genetic testing for thalassemia: experiences of multiple centers between 2019 and 2022.

In clinical practice, the success of preimplantation genetic testing for monogenic diseases (PGT-M) for thalassemia was hindered by the absence of probands, incomplete family members, or failure in detecting embryonic gene mutation sites. This study aimed to address these issues. This retrospective study included 342 couples undergoing PGT-M for α- or β-thalassemia at three reproductive medicine centers from 2019 to 2022. Various methods were used to construct parental haplotypes. A total of 1778 embryos were analyzed and selected for transfer based on chromosomal ploidy and PGT-M results. Follow-up involved amniocentesis results and clinical outcomes. Haplotypes were established using DNA samples from probands or parents, as well as sibling blood samples, single sperm, and affected embryos, achieving an overall success rate was 99.4% (340/342). For α-thalassemia and β-thalassemia, the concordance between embryo single nucleotide polymorphism (SNP) haplotype analysis results and mutation loci detection results was 93.8% (1011/1078) and 98.2% (538/548), respectively. Multiple annealing and looping-based amplification cycles (MALBAC) showed a higher whole genome amplification success rate than multiple displacement amplification (MDA) (98.8% (1031/1044) vs. 96.2% (703/731), p < 0.001). Amniocentesis confirmed PGT-M outcomes in 100% of cases followed up (99/99). This study summarizes feasible solutions to various challenging scenarios encountered in PGT-M for thalassemia, providing valuable insights to enhance success rate of PGT-M in clinical practice.

Successful development of molecular diagnostic technology combining mini-barcoding and high-resolution melting for traditional Chinese medicine agarwood species based on single-nucleotide polymorphism in the chloroplast genome.

Agarwood is a valuable traditional medicine and fragrance. The production process is a typical injury-induced defense response. Currently, there are approximately 22 known species in the genus Aquilaria Lam., all of which can produce agarwood, whereas there are only two legal species of traditional Chinese medicinal agarwood, Aquilaria sinensis (Lour.) Spreng. and Aquilaria agallocha (Lour.) Roxb. The Taiwan herbal Pharmacopoeia of China stipulates that the medicinal agarwood species are A. sinensis and its relatives in the same genus. Moreover, there are five species of agarwood available for clinical medicinal use in Japan, including A. agallocha and A. sinensis, which are often confused with each other or used in a mixed way in the trade process. Therefore, accurate identification of traditional Chinese medicinal agarwood species is important to ensure the authenticity of traditional medicines and to guide the safety of clinical medication. In this study, 59 specific single-nucleotide polymorphism loci were screened and obtained from the chloroplast genomes of 12 species of the genus Aquilaria Lam. We established an identification method for traditional Chinese medicinal agarwood using mini-barcoding combined with high-resolution melting (HRM) and designed and validated 10 pairs of primers from the psbM-trnD, psbA, rps16, petN, ndhE-psaC, rps4, atpE, ycf1, rps15-trnN, and matK regions. The amplification products were all less than 200 bp, with a high success rate of amplification. The method was applied to successfully identify traditional Chinese medicinal agarwood species from commercial agarwood samples. Overall, the sensitivity of this method was sufficient to detect 1% of adulterants in medicinal agarwood products, proving that mini-barcoding HRM is a powerful and flexible tool. This method can be used as a fast and effective high-throughput method for authenticity testing of traditional Chinese medicinal agarwood and its raw materials containing agarwood-containing proprietary Chinese medicines and is recommended for industrial applications.

Pupal Exuviae of Culex Pipiens L. (Diptera: Culicidae) Can be Utilised as a Non-Invasive Method of Biotype Differentiation

BackgroundCulex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay.ResultsGenomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55–80%.ConclusionsThis study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.

Comparing skin swabs, buccal swabs, and toe clips for amphibian genetic sampling, a case study with a small anuran (Acris blanchardi).

Multiple methods for collecting genetic samples from amphibians exist, each with their own implications for study design, animal welfare, and costs. Toe clipping is one common method, but there is ongoing debate regarding its potential detriment. Less invasive methods should be implemented, if efficacious, as amphibians are a particularly vulnerable vertebrate group. Skin and buccal swabbing are less invasive methods for genetic sampling, but the potential for contamination and a lower yield of DNA may exist. To compare these methods, we gathered skin swabs, buccal swabs, and toe clips from the same individuals of a relatively small anuran species, Blanchard's Cricket Frog (Acris blanchardi). We then compared DNA yield, DNA purity, amplification success rate, and genotypic data quality among sample types. We found toe clips and buccal swabs generated similar DNA yield and purity, with skin swabs yielding significantly less DNA of significantly lower purity than the other sample types. Amplification success rate was significantly higher using toe clips compared to the other sample types, though buccal swab samples amplified more readily than skin swabs. Genotypic data from toe clips and buccal swabs did not differ significantly in quality, but skin swab data quality was significantly lowest among sample types. Thus, skin swabbing could produce erroneous data in some situations, but buccal swabbing is likely an effective substitute to toe clipping, even for small species. Our results can help future researchers select which genetic sampling method might best suit their research needs.

Mammal dung-dung beetle trophic networks: an improved method based on gut-content DNA.

Dung beetles provide many important ecosystem services, including dung decomposition, pathogen control, soil aeration, and secondary seed dispersal. Yet, the biology of most dung beetles remains unknown. Natural diets are poorly studied, partly because previous research has focused on choice or attraction experiments using few, easily accessible dung types from zoo animals, farm animals, or humans. This way, many links within natural food webs have certainly been missed. In this work, we aimed to establish a protocol to analyze the natural diets of dung beetles using DNA gut barcoding. First, the feasibility of gut-content DNA extraction and amplification of 12s rDNA from six different mammal dung types was tested in the laboratory. We then applied the method to beetles caught in pitfall traps in Ecuador and Germany by using 12s rDNA primers. For a subset of the dung beetles caught in the Ecuador sampling, we also used 16s rDNA primers to see if these would improve the number of species we could identify. We predicted the likelihood of amplifying DNA using gut fullness, DNA concentration, PCR primer, collection method, and beetle species as predictor variables in a dominance analysis. Based on the gut barcodes, we generated a dung beetle-mammal network for both field sites (Ecuador and Germany) and analyzed the levels of network specificity. We successfully amplified mammal DNA from dung beetle gut contents for 128 specimens, which included such prominent species as Panthera onca (jaguar) and Puma concolor (puma). The overall success rate of DNA amplification was 53%. The best predictors for amplification success were gut fullness and DNA concentration, suggesting the success rate can be increased by focusing on beetles with a full gut. The mammal dung-dung beetle networks differed from purely random network models and showed a moderate degree of network specialization (H2': Ecuador = 0.49; Germany = 0.41). We here present a reliable method of extracting and amplifying gut-content DNA from dung beetles. Identifying mammal dung via DNA reference libraries, we created mammal dung-dung beetle trophic networks. This has benefits over previous methods because we inventoried the natural mammal dung resources of dung beetles instead of using artificial mammal baits. Our results revealed higher levels of specialization than expected and more rodent DNA than expected in Germany, suggesting that the presented method provides more detailed insights into mammal dung-dung beetle networks. In addition, the method could have applications for mammal monitoring in many ecosystems.

Development of a standardized methodology for selecting candidate DNA barcodes of Trillium govanianum to combat biodiversity crime and environmental exploitation

This study introduces an innovative method using DNA barcodes to accurately identify species, addressing the critical issue of trafficking invaluable medicinal plants like Trillium govanianum. The success rate in amplification and sequencing of barcoding regions was an impressive 100%. The Internal Transcribed Spacer (ITS) region proved most effective in distinguishing closely related Trillium species. By testing herbal products from markets, the study revealed extensive illegal trade, with 50% confirmed as Trillium govanianum and 30% adulterated with Dioscorea villosa. This research contributes new DNA sequences to identification databases, enhancing protocols. Genetic variations within Trillium govanianum were narrower than to the nearest non-conspecific neighbor in barcoding regions (matK: 0.006, rbcL: 0.003, ITS: 0.043). Combining coding and non-coding regions (matK + ITS) achieved 100% species discrimination. Market samples showed 30% product substitution, with varying success rates for amplification (rbcL: 70%, matK: 60%, ITS: 40%, improved with ITS2 primers). Half the sequences were Trillium govanianum, 30% were Dioscorea villosa, revealing covert substitution. ITS and matK sequences were effective, with a genetic distance gap, while rbcL had limited divergence (0.003), making it less suitable for accurate identification compared to matK and ITS.

Including the invisible fraction in whole population studies: A guide to the genetic sampling of unhatched bird eggs

Abstract Early embryo mortality has recently been proven to be a significant component of avian reproductive failure. Due to the difficulty in distinguishing eggs, which have suffered early embryo mortality from unfertilised eggs, this cause of reproductive failure has historically been underestimated and overlooked. We describe methods for recognising and collecting early failed, unhatched eggs from wild bird populations, identifying and isolating embryonic material in unhatched eggs, and efficiently extracting DNA from those samples. We test these methods on unhatched hihi (Notiomystis cincta) eggs collected from the field, which have undergone postmortem incubation. We obtained DNA yields from early‐stage embryos that are sufficient for a wide range of molecular techniques, including microsatellite genotyping for parentage analysis and sex‐typing. The type of tissue sample taken from the egg affected downstream DNA yields and microsatellite amplification rates. Species‐specific microsatellite markers had higher amplification success rates than cross‐species markers. We make key recommendations for each stage of the sampling and extraction process and suggest potential protocol improvements and modifications. Genetic and possibly genomic analysis of embryos that die early in development has the potential to advance many fields. The methods described here will allow a more in‐depth exploration of the previously overlooked causes of early embryo mortality in wild populations of birds, including threatened species.

UPrimer: A Clade-Specific Primer Design Program Based on Nested-PCR Strategy and Its Applications in Amplicon Capture Phylogenomics.

Amplicon capture is a promising target sequence capture approach for phylogenomic analyses, and the design of clade-specific nuclear protein-coding locus (NPCL) amplification primers is crucial for its successful application. In this study, we developed a primer design program called UPrimer that can quickly design clade-specific NPCL amplification primers based on genome data, without requiring manual intervention. Unlike other available primer design programs, UPrimer uses a nested-PCR strategy that greatly improves the amplification success rate of the designed primers. We examined all available metazoan genome data deposited in NCBI and developed NPCL primer sets for 21 metazoan groups with UPrimer, covering a wide range of taxa, including arthropods, mollusks, cnidarians, echinoderms, and vertebrates. On average, each clade-specific NPCL primer set comprises ∼1,000 NPCLs. PCR amplification tests were performed in 6 metazoan groups, and the developed primers showed a PCR success rate exceeding 95%. Furthermore, we demonstrated a phylogenetic case study in Lepidoptera, showing how NPCL primers can be used for phylogenomic analyses with amplicon capture. Our results indicated that using 100 NPCL probes recovered robust high-level phylogenetic relationships among butterflies, highlighting the utility of the newly designed NPCL primer sets for phylogenetic studies. We anticipate that the automated tool UPrimer and the developed NPCL primer sets for 21 metazoan groups will enable researchers to obtain phylogenomic data more efficiently and cost-effectively and accelerate the resolution of various parts of the Tree of Life.

Identification and diversity assessment of Ziziphus species using DNA barcoding

The genus Ziziphus, belonging to the family Rhamnaceae, is of great economic and medicinal importance worldwide due to the value of its fruits in household income and food security. However, many species within this genus are phenotypically identical, leading to imprecise identification and hindering their conservation, variation in the gene pool, and exploitation. To address this issue, we collected 26 individuals from different regions of Khyber Pakhtunkhwa, Pakistan and Shanxi province, China, to assess the efficiency of DNA barcoding regions in identifying Ziziphus species. We tested the ability of five DNA barcodes regions, namely ITS, matK, rbcL, trnH-psbA, and trnL-UAA, to distinguish between Ziziphus species as an alternative approach for species identification and the phylogenetic reconstruction. The results of the study, as the success rate of PCR amplification of matK, trnL-UAA, and ITS, provided the highest species resolution with 100 % accuracy, except for trnH-psbA (90.78 %). ITS and matK were identified as the most effective single barcode, with a species identification success rate of 87.4 %±0.98 % and 84.89 %±0.66 %. These results suggest that matK, trnL-UAA, and ITS are universal barcode regions for identification of all Ziziphus species identification and their discrimination power to differentiate. In contrast, the combination of trnL-UAA+ITS region displayed the highest species identification rate (92.1 %), the five barcodes region combination, and superior to any barcodes regions combination that included rbcL+trnH-psbA. Additionally, the study found high haplotypes and nucleotide diversity in Pakistani Ziziphus species, mainly in the ITS barcode, with values of 0.84 and 0.0086, respectively. In conclusion, the study establishes that the DNA fragments ITS, trnL-UAA, and matK are effective in identifying and distinguishing Ziziphus species. The approach can be considered as an accurate tool for identifying these species and will be used as a universal barcode's region for Ziziphus species. DNA barcoding is a useful approach and provides a reliable and effective means for Ziziphus discrimination, and in combination with morphology-based taxonomy will be a robust approach for tackling taxonomically complex species. Additionally, this study provides valuable insights into the genomic architecture of Ziziphus species, which has implications for their classification, conservation, and germplasm resource improvement. This research provides a novel approach to the problem of incorrectly identifying economically and medicinally important species, particularly those in the genus Ziziphus.

Evaluation of 20 CODIS Core STR Loci For Touch DNA Analysis

The DNA profile is a combination of STR alleles loci as a result of template DNA amplification. The FBI has defined 20 core STR loci for human identification in forensic cases. The aim of this study was to evaluate the twenty CODIS core STR loci to be a reference in DNA touch examination. Nine donors with three shedder statuses, high, intermediate, and low, were holding the sample while tying two types of ropes, porous and non-porous. The touch sample is then sampled using two techniques on each rope: double swab and tape lift. DNA was extracted and quantified by real-time PCR technique to determine the concentration of DNA obtained from each treatment. Twenty-four STR loci of the DNA were amplified, and the fragments were analyzed by capillary electrophoresis. Two PCR cycles were chosen to determine the effect of adding cycles on the concentration of DNA that was not successfully amplified. The results of this study indicate that the quantity of touch DNA has a greater influence on the success rate of STR amplification. Each allele on 20 STR CODIS Loci failed to amplify from touch DNA concentration below 0.0625 ng/µl. It is shown that the longer the target allele at each locus, the easier it is for allelic drop-out to occur. However, the optimal STR locus length for DNA testing is under 200 nucleotides.

Comprehensive analysis using DNA metabarcoding, SCAR marker based PCR assay, and HPLC unveils the adulteration in Brahmi herbal products.

Brahmi is one of the important nootropic botanicals, widely sold in the market, with the name "Brahmi'' being used to describe both Bacopa monnieri and Centella asiatica species. The Brahmi herbal products market is expanding; hence, economically motivated adulteration is highly prevalent. This study aimed to develop DNA-based methods, including SCAR marker-based PCR and metabarcoding, to authenticate Brahmi herbal products and compare these methods with HPLC. These methods have been validated using mock controls (in-house blended formulations). All targeted plant species in mock controls were detected successfully with all three methods, whereas, in market samples, only 22.2%, 55.6%, and 50.0% were found positive for Brahmi by PCR assay, DNA metabarcoding, and HPLC, respectively. Metabarcoding can detect the presence of non-labeled plants together with targeted species, which is an advantage over PCR assay or HPLC. SCAR marker-based PCR is a rapid and cost-effective method for detecting the presence of B. monnieri and C. asiatica. However, in this study, the success rate of PCR amplification was relatively low because the primers targeted either RAPD or ITS-based SCAR markers. HPLC assay, although an alternative, was unable to detect the presence of other botanicals, just like the SCAR marker-based PCR assay. On the other hand, metabarcoding can be utilized to identify the target plants, even in very small quantities, while also providing simulated identification of other botanicals. This study successfully addressed the need for quality control of Brahmi herbal products and provided the first-time report of DNA metabarcoding for such products.

Species identification of Ligustrum lucidum

Ligustrum lucidum is a woody perennial plant of genus Ligustrum in family Oleaceae. Its dried fruit has high medicinal value. In this study, the authors evaluated the variability and species identification efficiency of three specific DAN barcodes(rbcL-accD, ycf1a, ycf1b) and four general DAN barcodes(matK, rbcL, trnH-psbA, ITS2) for a rapid and accurate molecular identification of Ligustrum species. The results revealed that matK, rbcL, trnH-psbA, ITS2 and ycf1a were inefficient for identifying the Ligustrum species, and a large number of insertions and deletions were observed in rbcL-accD sequence, which was thus unsuitable for development as specific barcode. The ycf1b-2 barcode had DNA barcoding gap and high success rate of PCR amplification and DNA sequencing, which was the most suitable DNA barcode for L. lucidum identification and achieved an accurate result. In addition, to optimize the DNA extraction experiment, the authors extracted and analyzed the DNA of the exocarp, mesocarp, endocarp and seed of L. lucidum fruit. It was found that seed was the most effective part for DNA extraction, where DNAs of high concentration and quality were obtained, meeting the needs of species identification. In this study, the experimental method for DNA extraction of L. lucidum was optimized, and the seed was determined as the optimal part for DNA extraction and ycf1b-2 was the specific DNA barcode for L. lucidum identification. This study laid a foundation for the market regulation of L. lucidum.

HIV-1 RNA and DNA Genotyping Drug Resistance Detection in Patients with Low-Level Viremia in Liangshan, China.

In this study, we characterized HIV-1 RNA and HIV-1 DNA genotyping drug resistance detection in patients with low-level viremia (LLV) in Liangshan, China. Whole blood samples were collected from HIV/AIDS patients who had received antiretroviral therapy (ART) for ≥6 months and whose HIV-1 RNA loads were 50-1,000 copies/mL for two consecutive times at least 1-month apart. The patients were enrolled from a county in Liangshan Yi Autonomous Prefecture, Sichuan Province, between May 2021 and May 2022. Plasma and blood cells were separated. Plasma samples were tested for HIV-1 RNA genotyping drug resistance, while blood cell samples were tested for HIV-1 DNA genotyping drug resistance. Then, HIV-1 RNA and HIV-1 DNA genotyping drug resistance outcomes were compared. Among the 32 participants, 16 were males, while 16 were females, with the median age of 34.5 years. The main HIV-1 infection route was heterosexual transmission. The median ART duration was 3.9 years. Two types of nucleoside reverse transcriptase inhibitors (NRTIs) + one non-nucleoside reverse transcriptase inhibitor (NNRTI) were the main antiviral therapeutic options. Pol region genes for 28 HIV-1 DNA samples and 10 HIV-1 RNA samples were successfully amplified. The success rate of pol region gene amplification for HIV-1 DNA was significantly higher than that of HIV-1 RNA (χ2 = 20.988, p < .05). In HIV-1 RNA and HIV-1 DNA samples, M184 (4/8) and K103 (3/8) were the most frequent drug resistance mutation sites. Among the NNRTIs, the rates of drug resistance were highest to efavirenz (EFV) (6/8) and nevirapine (NVP) (6/8), while among the NRTIs, the rates of drug resistance were highest to abacavir (ABC) (4/8), emtricitabine (FTC) (4/8), and lamivudine (3TC) (4/8). In conclusion, detection of HIV-1 RNA genotyping drug resistance combined with HIV-1 DNA genotyping drug resistance can improve the success rate of drug resistance detection in patients with LLV.

In Vitro Isolation and Molecular Characterization of Genomic DNA from Ancient Human Long and Hip Bones

Background: A simple and effective modified ethanol precipitation-based protocol was described for the extraction of genomic DNA from ancient human bones. The qualitative and quantitative evaluation of genomic DNA was done based on DNA purity (260/280) and the PCR method. Method and Materials: In this study, a total of 50 embalmed ancient bones, including 20 long and 30 hip bone samples, were taken for genomic DNA extraction. The efficiency of the genomic DNA extraction was demonstrated on &gt;50-year-old ancient human HIP and long bone samples. In vitro quantitative and qualitative analysis of extracted genomic DNA was performed by 0.8% agarose gel electrophoresis and PCR amplification. To assess the quality of extracted genomic DNA, a mitochondrial-specific ATPase6 gene primer was used to obtain sequence information of 675 bp. Result: Our data show that a concentration of genomic DNA between 1.6 and 2.0 at 260/280 resulted in successful PCR amplification. Our results demonstrated that the extraction of DNA from ancient bone samples with a manual approach will increase the amplification efficiency of the polymerase chain reaction (PCR). Conclusion: In the present study, a simple, time-saving, and cost-effective protocol is described for the extraction of genomic DNA from ancient human bones. Further, we believe the extraction of genomic DNA from ancient bone samples with this approach will increase the success rate of PCR amplification.

Resistance levels to non-nucleoside reverse transcriptase inhibitors among pregnant women with recent HIV infection in Malawi.

Background:Information on HIV drug resistance (HIVDR) prevalence in people newly diagnosed with HIV is limited. We implemented a cross-sectional study to estimate HIVDR prevalence among pregnant women recently infected with HIV in Malawi.Methods:The HIVDR study was nested within a routine antenatal clinic (ANC) sentinel surveillance survey. Dried blood spot samples were tested for recent infection using a limiting antigen antibody assay together with HIV viral load testing. HIV-1 protease and reverse transcriptase were sequenced using Sanger sequencing. Drug susceptibility was predicted using Stanford HIVdb algorithm (version 8.9). Weighted analysis was performed in Stata 15.1.Results:Of the 21,642 pregnant women enrolled in the ANC survey, 8.4% (1826/21,642) tested HIV positive. Of these, 5.0% (92/1826) had recent HIV infection, and 90.2% (83/92) were tested by PCR. The amplification and sequencing success rate was 57.8% (48/83). The prevalence of any HIVDR was 14.6% (5/45) (95% CI: 4.7–36.8%), all of which indicated HIVDR to nonnucleoside reverse transcriptase inhibitors (NNRTIs). HIVDR to nucleoside reverse transcriptase inhibitors was 7.9% (2/45) (95% CI: 1.4–34.6%). Resistance to protease inhibitors currently in use in Malawi was not observed.Conclusions:Despite the low number of cases with presumed TDR, our study hints that resistance to NNRTIs was high, above the 10% target for regimen change. Further investigation is needed to establish the exact magnitude of presumed TDR among women recently infected with HIV. These findings support the transition to an integrase inhibitor-based first-line regimen for patients initiating or on ART.

Assessing the utility of quantitative and qualitative metrics in the DNA quantification process of skeletal remains for autosomal and Y-chromosome STR amplification purposes

In historical cases, ancient DNA investigations and missing persons identification, teeth or bone samples are often the only and almost always the best biological material available for DNA typing. On the other hand, DNA obtained from bone material may be characterized by a high degradation index (DI) or its low content, or DNA tests cannot be repeated due to bone piece size limitation. That is often the effect of the environment in which the material was placed and the time during which exposure to unfavorable environmental factors took place. Therefore, it is very important to use appropriate procedures related to STR analysis. For our study, we selected 80 challenging bone samples. The amount of DNA was compared in qPCR using Quantifiler™ Trio DNA Quantification Kit and Investigator® Quantiplex® Pro RGQ. All qPCR results were confirmed by PCR-CE. The results of DNA concentrations and the assigned degradation index (DI) differed significantly within analyzed samples (~10%). Additionally, the Y-chromosome DI also differed from the autosomal DI in the samples. The difference in degradation indexes could explain the lower Y-chromosome amplification success rate compared to autosomal e.g. during human identification process. The results indicate that performing two DNA quantifications with the use of two different kits (primers sets) allows for a much more precise evaluation of the DNA quality and quantity in the isolate. We suggest that at least one of two suggested DNA concentration measurements should be based on an additional determination of the Y chromosome degradation index. Altogether, it allows for rational isolate management, especially when the volume is limited and the sample is unique.

Molecular identification and phylogenetic relationships of Colletotrichum isolates pathogenic on cultivated plants in Bulgaria

Fungi of the genus Colletotrichum are causal agents of plant diseases with constantly growing economic importance. Accurate pathogen identification is a significant prerequisite for effective disease control. The aim of the present investigation was to clarify the species affiliation of Colletotrichum isolates obtained from different hosts in Bulgaria and to determine the phylogenetic relationships between them by applying DNA barcoding. Thirty-five fungal isolates obtained from five botanical families (Solanaceae, Rosaceae, Musaceae, Cucurbitaceae and Caryophyllaceae) were morphologically characterized and subjected to molecular analysis based on four fungal barcode markers – the primary ITS barcode and the secondary marker regions ACT, EF-1a and TUB2 (Fig. 1). Three of the barcodes (ITS, ACT and TUB2) showed complete success rate of PCR amplification and sequencing and proved efficient for reliable identification at species level. BLAST analyses identified eleven Colletotrichum species assigned to five different complexes – C. coccodes, C. acutatum, C. gloeosporioides, C. dematium and C. spaethianum. The resolution power of ITS region was not sufficient to discriminate interspecies variations within C. coccodes, C. dematium and C. spaethianum complexes confirming the requirement for secondary barcodes in order to resolve the genetic variability of the Colletotrichum isolates. DNA barcoding analyses revealed that the highest species variation was observed among the isolates from pepper (Capsicum annuum). Interestingly, an isolate from the same host identified as C. truncatum on the basis of morphological characters appeared to be C. circinans when applying DNA barcode markers. According to our knowledge, this species has not been reported as a causal agent of pepper anthracnose. Data obtained in this study improve our understanding of the genetic diversity within the Colletotrichum population pathogenic on cultivated plants in Bulgaria.

DNA metabarcoding unravels unknown diversity and distribution patterns of tropical freshwater invertebrates

Abstract Tropical freshwater invertebrate species are becoming extinct without being described, and effective conservation is hampered by a lack of taxonomic and distribution data. DNA metabarcoding is a promising tool for rapid biodiversity assessments that has never been applied to tropical freshwater invertebrates across large spatial and taxonomic scales. Here we use DNA metabarcoding to comprehensively assess the benthic freshwater invertebrate fauna of the Perak River basin, Malaysia. Specific objectives were to: (1) assess performance of two DNA metabarcoding protocols; (2) identify gaps in reference databases; (3) generate new data on species diversity and distribution; and (4) draw conclusions regarding the potential value of DNA metabarcoding in tropical freshwater conservation. Organisms were collected by hand and net at 34 sites and divided into small (retained in 0.5‐mm but passing through 1‐mm mesh) and large (retained in 1‐mm mesh) fractions, and a 313‐bp cytochrome c oxidase subunit I fragment amplified and sequenced using general Metazoa primers. Bioinformatic analysis resulted in 468 operational taxonomic units (~species) from 12 phyla. Only 29% of species could be assigned binominal names through matches to public sequence libraries, indicating varying levels of library completeness across Orders. Extraction of small‐fraction DNA with a soil kit resulted in a significantly higher species count than with a general kit, but this was not even across taxa. Metabarcoding (amplification) success rate, estimated via comparison to morphological identifications of the large‐fraction specimens, was high in most taxa analysed but low, for example, in ampullariid and viviparid gastropods. Conversely, a large proportion of species‐site records for Decapoda and Bivalvia came from metabarcoding only. Species richness averaged 29 ± 16 species per site, dominated by Diptera, Annelida, and Odonata, and was particularly high in tributaries of the mountainous Titiwangsa Range. At least eight species are new records for Malaysia, including the non‐natives Ferrissia fragilis (Gastropoda) and Dugesia notogaea (Platyhelminthes). Our study showed that DNA metabarcoding is generally more effective in detecting tropical freshwater invertebrate species than traditional morphological approaches, and can efficiently improve knowledge of distribution patterns and ranges of native and non‐native species. However, current gaps in reference databases, particularly for bioindicator taxa, such as the Plecoptera, Ephemeroptera, and Coleoptera, need to be addressed urgently.

Character of cell-free genomic DNA in embryo culture medium and the prospect of its clinical application in preimplantation genetic testing

Abstract There is increasing evidence that cell-free DNA (cfDNA) in spent culture media (SCM) can be amplified for genetic testing. Therefore, this paper reviews the characteristics of cfDNA, including its fragment size, amount, origin, as well as some factors affecting the success rate of its amplification, together to provide researchers with a more comprehensive perspective on embryonic cfDNA. The origin of cfDNA in SCM is complicated and poses challenges to the interpretation of genetic test results. Advanced molecular techniques should distinguish between embryonic and contaminated DNA to maximize the success rate of amplification and analysis. Recent data showed that the type of culture medium, assisted hatching or not, the type of amplification kit, and fresh or thawed embryos were not related to the success rate of amplification, but the length of culture time might affect the success rate. The longer culture time, the more cfDNA is available in the SCM. Then we focused on the concordance between trophectoderm (TE), inner cell mass, whole embryo, and embryonic cfDNA. Despite successful amplification, the concordance between TE and embryonic cfDNA was low. In summary, non-invasive genetic testing using SCM could represent a major advance in future single embryo selection, however, contamination and timing for media collection are key factors affecting the results, and current noninvasive cfDNA testing should not be directly applied to clinical practice. Further research is needed to improve the methods used for testing techniques and genetic analysis to achieve greater accuracy and trace its origins before it can be used in the clinics.