PCR-amplified DNA Research Articles

First Report of Cladosporium ramotenellum Causing Sooty Spot on Postharvest Clementines in the United States.

In 2022, post-harvest symptoms of black spots were observed at an incidence of 2-5% on easy peeling clementines (citrus × clementina) in a fresh fruit market in Phoenix metropolitan area, Arizona. Black lesions on the fruit rind were superficial, circular, dry, and firm with gray sporulation. Gray or black aerial mycelium was also noticed on top of the black spots. Black spots were distributed over the entire fruit surface without any regular patterns. Maceration of rind and flesh was also noticed under refrigerated storage conditions. Two isolates were obtained (S13 and S14) and cultured on potato dextrose agar (PDA) at 25oC for 14 days. The colony color and texture of both isolates were identical on PDA: olivaceous black with abundant sporulation, margin entire edge to slightly undulate. Conidia were 0-1 septate and globose or ellipsoid with sizes ranging from 4-10 × 2-4 μm (n = 15). Ramoconida were also 0-1 septate and ellipsoid to cylindrical with sizes ranging from 15-30 × 2-5 μm (n = 15). Conidiophore had a width of 3-6 μm (n = 15). Isolates were identified as Cladosporium ramotenellum based on these morphological features (Bensch et al. 2012). The IDs of these two isolates were further confirmed by genomic DNA isolation, PCR amplification and sequencing of the ITS1-5S-ITS2-28S region of rDNA (V9G/LR5 de Hoog and Gerrits van den 1998, primers ITS4/ITS5 White et al.), actin gene (ACT-512F/ACT-783R, Carbone and Kohn 1999), and elongation factor 1α gene (EF1-1018F/EF1-1620R, Stielow 2015). Sequences of the two isolates were identical and thus only one sequence of each gene was deposited in the GenBank. A BLASTn search of actin sequence (233-bp, OQ185511) revealed 99.1% match with ex-type sequence EF 679538 (strain CBS:121628) of C. ramotenellum holotype (query coverage: 97%). BLASTn analysis of a portion of EF-1α gene (579-bp, OQ185512) revealed more than 99.7% similarity with sequences KU933429 (ATCC strain 16022) and MT881827 (strain 18EPLE003) of C. ramotenellum (query coverage: 100%). The ITS sequence (1519-bp, OQ236707) was identical to the ITS sequences of C. ramotenellum strains in easy peeler mandarins from Peru (Murciano et al. 2021). Pathogenicity tests were carried out twice on fresh easy-peeling clementine fruit. The inocula (1 x 105 spores/ml) were prepared in sterile distilled water containing 0.1% Tween 20 (TW) by mixing the conidial suspensions of two isolates from 7-day-old PDA cultures. Ten fruit were washed, surface sanitized with 70% ethanol, and wound-inoculated by immersing five fruit in spore suspension for 1 min. Five control fruit were wound-inoculated with TW. Inoculated and control fruit were stored in separate zip-lock bags for one week. Black spots resembling those observed on naturally infected fruit were present on inoculated fruit, while control fruit remained symptomless. C. ramotenellum was reisolated and was morphologically identical to the original isolates, thus completing Koch's postulates. C. ramotenellum has been reported as a fungal pathogen causing sooty spots on easy peeler mandarins from Peru (Murciano et al. 2021). To our knowledge, this is the first report of C. ramotenellum causing postharvest sooty spot and decay on clementines in the United States. Results show that infected fruit is potentially a pathogen source for long-distance dispersal. This occurrence was communicated to the state regulatory agencies for regulatory actions on imports of citrus fruit from countries with the occurrence of C. ramotenellum.

Association of g.331T>C and g.1909T>C locus in KISS1 gene with fecundity traits of Kacang and Boerka goats

Abstract. Maskur, Jan R, Lestari, Rozi T, Muhsinin M. 2023. Association of g.331T>C and g.1909T>C locus in KISS1 gene with fecundity traits of Kacang and Boerka goats. Biodiversitas 24: 4870-4876. The neuropeptide kisspeptin encodes the KISS1 gene and is crucial for reproductive system signals. This study aimed to identify polymorphisms of fecundity genes by observing candidate genes for prolific traits, namely the KISS1 gene (exon 1 and intron 1), and their relationship with litter size in Kacang and Boerka goats (Capra aegagrus hircus). The total sample used was 211 female goats. The stages of the research included DNA extraction, PCR amplification and genotyping. The association of KISS1 gene genotypes with litter size traits in Kacang goats and Boerka was analyzed using the SAS 9.1.3 software with General Linear Model (GLM) procedure. The results showed that the g.331T>C locus of the KISS1 exon 1 gene was monomorphic in all Kacang and Boerka goats populations. The locus g.1909T>C of the KISS1 intron 1 gene was polymorphic in all Kacang and Boerka goats populations. The locus g.1909T>C of the KISS1 intron 1 gene found three genotypes (TT, AT, AA). The frequency of allele T (0.775 and 0.815) was higher than allele A (0.225 and 0.185) in all Kacang and Boerka goats populations. The association of TT and AT genotypes with litter size significantly differed from that of the AA genotype in the Kacang goat. Meanwhile, in the Boerka goat, the TT genotype significantly differed from the AT and AA genotypes. The goat KISS1 gene polymorphism is critical to reproductive function and may be a reproductive characteristic marker.

Investigation of hepatitis B virus mutations associated with immune escape and drug resistance in human immunodeficiency virus-infected patients

Background: HBV/HIV co-infection impact on high HBV replication, progression to liver cancer and high mortality. Co-infection may lead to cross-resistance of HBV and HIV drugs due to immune therapy pressure or hepatotoxicity. These challenges necessitate continuous monitoring of HBV variants to aid better diagnosis and treatment strategies. We conducted this study to characterise HBV among HIV infected individuals. Methods: Serum was screened for HBsAg using ELISA, followed by DNA extraction, PCR amplification, Sanger sequencing and phylogenetic analysis. Results: Of the 50 samples in this study 100% (N=50/50) were HBsAg positive; 78% (N=41/50) HBV/HIV co-infection and 92% (N=38/41) of the amplicons were successfully sequenced. Samples nucleotide sequences were identified as genotype A. Mutations prevalence in the HBsAg region was 47% (N=18/38); including mutations associated with diagnostic failure (K122R and T143S) and 7 vaccines escape mutations (P127T, G145R, S207N, Y200T, E164D, Y206H and L209V). Mutations showed resistance to lamivudine 71% (n=5/7), telbivudine 57% (n=4/7), 14% (n=1/7) for entecavir and 43% (n=3/7) for adefovir. Mutations causing resistance to lamivudine and telbivudine were M204V, L180M, V163I, and S202K; with S202K also causing resistance to entecavir and adefovir resistance mutation were I253Y, I223V and M250I. Multiple drug resistance mutations within a single sample contained L180M, M204V, S202K and M250I mutations. There was no statistical significance between the RT mutations associated with drug resistance at P>0.005. The correlation test exhibited a weak statistical association between SHB and RT mutations (0.877**). Conclusions: This study shows the predominance of HBV genotype A in HIV-infected patients. We discovered HBV mutations linked to immune evasion and drug resistance. Although there is no statistical significance amongst the mutations associated with drug resistance and vaccine escape. These mutations could have clinical implications that could have therapeutic repercussions by influencing the correct clinical diagnosis and treatment in HBV/ HIV co-infected individuals.

B-217 The study of association of FokI Polymorphism of Vitamin D Receptor (VDR) Gene, Vitamin D and Parathyroid Hormone Levels in Stage 4–5 Chronic Kidney Disease Patients: A Cross-sectional Study

Abstract Background Chronic Kidney Disease (CKD) is an important public health problem globally and almost 35% patients of CKD have deficiency in vitamin D which progresses to 80% in stage 4–5 CKD patients. Active vitamin D binds to Vitamin D Receptor (VDR) to regulate target gene transcription. Various genetic variations (Single Nucleotide Polymorphism) such as FokI polymorphism, which is a T/C transition polymorphism (ACG to ATG) may affect the VDR gene expression and thus may play an important role in pathogenesis of CKD. The aim of the study is to study association between FokI Polymorphism of Vitamin D Receptor (VDR) Gene, serum Vitamin D and PTH levels in Stage 4–5 CKD patients. Methods A total of 150 patients, aged 25–60 years, attending the Department of Nephrology AIIMS New Delhi with chronic kidney disease (CKD) stage 4–5, were enrolled for the study. FokI polymorphism was analyzed by using polymerase chain reaction-restriction fragment length Polymorphism (PCR-RFLP) in study subjects. Following DNA isolation and PCR amplification, restriction digestion was done using FokI restriction endonuclease and visualised on 1.5 % agarose gel electrophoresis. The prevalence of different genotypes and allelic frequency distribution was compared between CKD patients and healthy controls (HC). Levels of PTH, Vitamin D were estimated by eCLIA. Results No significant differences in genotype and in allelic frequencies between CKD patients and HC were observed as shown in Table 1. No significant differences in biochemical parameters based on genotypic variations in CKD patients and in HC were observed. Conclusion The present study reveals no association of VDR FokI polymorphism with CKD. Furthermore, no significant differences in biochemical parameters were observed in FF, Ff and ff genotypic subgroups in CKD or HC groups. Further analysis revealed no significant differences in biochemical parameters based on genotypic variations in CKD patients and in HC. Table 1.Genotype frequency and allelic frequency of VDR FOK1 gene polymorphism in CKD patients and HC.Genotype(FOK1)Genotype frequency (%)P-valueX2 (chi2)Allelic frequencyCKD (n = 150)HC (n = 150)Total study group (n = 300)0.7700.5220CKD (n = 150)HC (n = 150)FF53.33 (n = 80)57.33 (n = 86)55.33 (n = 166)FfFfFf41.33 (n = 62)37.33 (n = 56)39.33 (n = 118)0.740.260.760.24ff5.33 (n = 8)5.33 (n = 8)5.33 (n = 16)

Portuguese Neonatal Screening Programme: A Retrospective Cohort Study of 18 Years of MS/MS.

The Portuguese Neonatal Screening Programme (PNSP) identifies patients with rare diseases through nationwide screening. Currently, 27 diseases are diagnosed, amongst which are 24 Inborn Errors of Metabolism (IEM), covering approximately 100% of neonates (1). In 2004, the national laboratory implemented a new screening method, tandem mass spectrometry (MS/MS) to test for amino acids and acylcarnitines. This new protocol revolutionized the PNSP and allowed for the analysis of an increased number of IEM, with clear improvements in treatment timings and clinical outcomes (2). From 2004 to 2022, 1 764 830 neonates were screened with MS/MS technology. Those who displayed biochemical profiles indicating an IEM were subjected to molecular characterization via genomic DNA extraction, PCR amplification, and direct Sanger sequencing method of dried blood spot samples. A cohort of 681 newborns were diagnosed with an IEM. MCAD deficiency is the most frequent, with 233 confirmed diagnoses, showing predominantly c.985A>G (p.K329E) mutation of the ACADM gene in homozygosity. Approximately 1/3 of the 33 confirmed cases of Glutaric Aciduria type I present homozygous for the c.1204C>T (p.Arg402Trp) mutation in GCDH. Around 60% of cases of MAT II/III deficiency display the dominant mutation of the MAT1A gene, c.791G>A (p.Arg264His). These genetic profiles and others were determined as diagnostic confirmation for 24 of the IEM screened. This data shows the molecular epidemiology of patients with confirmed IEM diagnosis identified by neonatal screening. Some diseases out of the scope of the PNSP were also detected as a differential diagnosis after biochemical suspicion in the dried blood spot sample. The retrospective analysis of the PNSP allows for an overview of 18 years of achievements accomplished by the national screening for IEM since MS/MS was implemented. For some pathologies with low incidence, it's difficult to trace a discernible pattern. However, presenting de novo mutations for these diseases might provide insights on how to approach different phenotypes. The aim of this work is to establish the molecular epidemiology of metabolic diseases screened.

Impact of Experimental Bias on Compositional Analysis of Microbiome Data.

Microbiome data are subject to experimental bias that is caused by DNA extraction and PCR amplification, among other sources, but this important feature is often ignored when developing statistical methods for analyzing microbiome data. McLaren, Willis, and Callahan (2019) proposed a model for how such biases affect the observed taxonomic profiles; this model assumes the main effects of bias without taxon-taxon interactions. Our newly developed method for testing the differential abundance of taxa, LOCOM, is the first method to account for experimental bias and is robust to the main effect biases. However, there is also evidence for taxon-taxon interactions. In this report, we formulated a model for interaction biases and used simulations based on this model to evaluate the impact of interaction biases on the performance of LOCOM as well as other available compositional analysis methods. Our simulation results indicate that LOCOM remained robust to a reasonable range of interaction biases. The other methods tend to have an inflated FDR even when there were only main effect biases. LOCOM maintained the highest sensitivity even when the other methods could not control the FDR. We thus conclude that LOCOM outperforms the other methods for compositional analysis of microbiome data considered here.

Correction: Sha et al. Differences in Root Endophytic Bacterial Communities of Chinese Cork Oak (Quercus variabilis) Seedlings in Different Growth Years. Forests 2023, 14, 1489

There was an error in the last sentence in part “2.1. Study Area and Sample Collection”, and the first sentence in “2.3. DNA Extraction and PCR Amplification” in the original publication [...]

TFOFinder: Python program for identifying purine-only double-stranded stretches in the predicted secondary structure(s) of RNA targets.

Nucleic acid probes are valuable tools in biology and chemistry and are indispensable for PCR amplification of DNA, RNA quantification and visualization, and downregulation of gene expression. Recently, triplex-forming oligonucleotides (TFO) have received increased attention due to their improved selectivity and sensitivity in recognizing purine-rich double-stranded RNA regions at physiological pH by incorporating backbone and base modifications. For example, triplex-forming peptide nucleic acid (PNA) oligomers have been used for imaging a structured RNA in cells and inhibiting influenza A replication. Although a handful of programs are available to identify triplex target sites (TTS) in DNA, none are available that find such regions in structured RNAs. Here, we describe TFOFinder, a Python program that facilitates the identification of intramolecular purine-only RNA duplexes that are amenable to forming parallel triple helices (pyrimidine/purine/pyrimidine) and the design of the corresponding TFO(s). We performed genome- and transcriptome-wide analyses of TTS in Drosophila melanogaster and found that only 0.3% (123) of total unique transcripts (35,642) show the potential of forming 12-purine long triplex forming sites that contain at least one guanine. Using minimization algorithms, we predicted the secondary structure(s) of these transcripts, and using TFOFinder, we found that 97 (79%) of the identified 123 transcripts are predicted to fold to form at least one TTS for parallel triple helix formation. The number of transcripts with potential purine TTS increases when the strict search conditions are relaxed by decreasing the length of the probe or by allowing up to two pyrimidine inversions or 1-nucleotide bulge in the target site. These results are encouraging for the use of modified triplex forming probes for live imaging of endogenous structured RNA targets, such as pre-miRNAs, and inhibition of target-specific translation and viral replication.

Assessing the Bioreceptivity of Biobased Cladding Materials

Materials exposed to the outdoors are prone to various deterioration processes. Architectural coatings are designed to protect surfaces against environmental and biotic degradation and to provide a decorative layer. The objective of this work was to examine the early colonisers on a diverse set of coated and non-coated biobased façade materials. A set of 33 wood-based cladding materials were exposed to four cardinal directions and monitored in outdoor conditions. The surfaces were sampled using a wet swab and plated on DG-18 agar, which prevents the growth of bacteria and limits the growth of fast-growing fungi. Pure cultures were then isolated and identified through PCR amplification and Sanger sequencing of specific DNA regions/genes. The response of cladding materials to weathering and fungal infestation was assessed. The proposed techniques enabled the identification of features that promote/inhibit fungal colonisation and revealed the preference of certain fungi for specific materials. Both the material type and the climate condition at the exposure site influence fungal colonisation. This study is a starting point for more exhaustive assays that aim to develop a novel coating system based on controlled and optimized fungal biofilm formation, and is proposed as a nature-inspired alternative for the protection of architectonic surfaces.

Impact of active lifestyle on the primary school children saliva microbiota composition.

Male (114) and female children (8-10 years) belonging to five primary schools in the neighborhoods of Turin were classified as active (A) or sedentary (S) based on PAQ-C-It questionnaire. PCR amplification of salivary DNA targeted the hypervariable V3-V4 regions of the 16S rRNA bacterial genes. DADA2 workflow was used to infer the Amplicon Sequence Variants and the taxonomic assignments; the beta-diversity was obtained by PCoA with the UniFrac method; LEfSe algorithm, threshold at 5%, and Log LDA cutoff at ±0.5 were used to identify differently abundant species in A compared to S saliva sample. Daily food intake was assessed by 3-Days food record. The metabolic potential of microbial communities was assessed by PICRUSt. No significant differences were found in individual's gender distribution (p = 0.411), anthropometry, BMI (p > 0.05), and all diet composition between A and S groups (p > 0.05). Eight species were differently abundant: Prevotella nigrescens (LDA score = -3.76; FDR = 1.5×10-03), Collinsella aerofaciens (LDA score = -3.17; FDR = 7.45×10-03), Simonsiella muelleri (LDA score = -2.96; FDR = 2.76×10-05), Parabacteroides merdae (LDA score = -2.43; FDR = 1.3×10-02) are enriched in the A group; Gemella parahaemolysans, Prevotella aurantiaca (LDA score = -3.9; FDR = 5.27×10-04), Prevotella pallens (LDA score = 4.23; FDR = 1.93×10-02), Neisseria mucosa (LDA score = 4.43; FDR = 1.31×10-02; LDA score = 2.94; FDR = 7.45×10-03) are enriched in the S group. A prevalence of superpathway of fatty acid biosynthesis initiation (E. coli) and catechol degradation II (meta-cleavage pathway) was found in saliva from A compared to S children. Our results showed that active children had an enrichment of species and genera mainly associated with a healthier profile. By contrast, the genera and the species enriched in the sedentary group could be linked to human diseases.

Development of a PCR-based lateral flow immunoassay for the identification of rainbow trout ingredient in foods

A simple PCR-based lateral flow immunoassay (LFIA) was developed in this study to identify rainbow trout ingredient in foods. This assay was developed by coupling PCR amplification of rainbow trout DNA with the detection of labeled amplicon using LFIA. The rainbow trout-specific PCR primers targeted at mitochondrial ATP6 gene were successfully designed. The color signals of LFIA could be observed by the naked eye within 5 min. The reliability of this assay was confirmed by quantitative real-time PCR (qPCR). The proposed assay could achieve an LOD at 0.1 ng/μL of rainbow trout DNA. The specificity of the primers was validated against 17 other species related to fish meat fraud and no cross-reactivity was identified, followed by successful test of commercial products for fish meat authentication. PCR-LFIA is simpler and faster than the conventional qPCR and has great potential to be applied to rapidly authenticate rainbow trout ingredient in foods.

Experimental infection reveals mud crab, Scylla serrata is less susceptible than Scylla olivacea and shrimp, Penaeus vannamei to white spot syndrome virus (WSSV)

White spot syndrome virus (WSSV) causes severe mortality in farmed shrimps, besides causing infection in a wide range of crustaceans. Among the hosts, mud crabs are reported to be relatively resistant to the virus. To validate this, the present study investigated the susceptibility of two species of mud crabs, Scylla olivacea and S. serrata compared with shrimp, Penaeus vannamei to WSSV through experimental infection. Initially, to find out the pathogenicity of the virus, a quantified, 10-fold serially diluted WSSV inoculum was injected intramuscularly into shrimp and mud crabs. Cumulative mortality of 100% was observed in shrimp injected with 10−1 and 10−2 WSSV dilution at 5 and 6 day post-injection (dpi), respectively. However, injection of the virus inoculum of 10−1 dilution resulted in 100% and 85% cumulative mortality at 7 dpi in S. olivacea and S. serrata, respectively. The LD50 (lethal dose 50% endpoint) value of WSSV in P. vannamei, S. olivacea and S. serrata was determined to be 1.74 × 105, 3.80 × 106 and 8.51 × 106 viral copies per animal, respectively. PCR amplification of DNA extracted from different tissues of the experimental animals at 24, 48 and 72 h post-injection (hpi) confirmed the infection. A SYBR green-based real-time PCR assay revealed comparatively higher viral load in shrimp tissues (4.1 × 103, 2.8 × 106 and 4.3 × 107 copies/ 100 ng DNA in stomach, 1.0 × 103, 2.4 × 106 and 3.5 × 107 copies/ 100 ng DNA in gill and 3.5 × 102, 1.1 × 104 and 1.1 × 105 copies/ 100 ng DNA in hepatopancreas at 24, 48 and 72 h, respectively). The viral load in S. olivacea was estimated to be 2.9 × 103, 2.2 × 106 and 3.0 × 107 copies/ 100 ng DNA in the stomach, 7.1 × 102, 1.6 × 106 and 2.4 × 107 copies/ 100 ng DNA in gill and 2.0 × 102, 8.6 × 103& 7.8 × 104 copies/ 100 ng DNA and in S. serrata the viral copy numbers detected were 1.1 × 103, 1.7 × 106 and 1.8 × 107 copies/ 100 ng DNA in stomach, 6.6 × 102, 1.3 × 106 and 1.4 × 107 copies/ 100 ng DNA in gill and 5.8 × 101, 7.7 × 102 & 1.1 × 104 copies/ 100 ng DNA at 24, 48 and 72 h, respectively. A higher viral load was detected in the stomach tissue compared to the gill and hepatopancreas of all the experimental animals. Histological examination of the stomach and gill of P. vannamei, S. olivacea and S. serrata at different time points post-infection revealed characteristic WSSV pathology. Hepatopancreatic tubular epithelium showed no viral inclusions, though pathological changes could be noticed in the intertubular connective tissue. The most pronounced changes were found in the stomach epithelium of P. vannamei and S. olivacea, which revealed significant nuclear hypertrophy and large-sized inclusions, compared to S. serrata which showed relatively smaller-sized inclusion bodies.

First report of rootstalk rot of Hibiscus mutabilis caused by Fusarium oxysporum in China.

H. Mutabilis (Cotton rose or confederate rose) is a deciduous shrub in the Malvaceae family, with ornamental, medicinal and edible values (Fan et al. 2015). In May to August 2020, 40.4% of potted plants of H. mutabilis were found to have root and stalk rot in Chengdu Botanical Garden (E104°7'28″, N30°45'57″). At first the leaves of affected H. mutabilis turned yellow and wilted, followed by the stem and root cortex became dark brown and rotten. Finally, the whole plant died within two months. Root and stem produced white mycelium when the humidity exceeded 90%. Samples taken from the lesions were surface disinfested for 3 min in 4% sodium hypochlorite, rinsed in sterile water and plated on potato dextrose agar (PDA), 35 single-spore cultures with similar morphology isolated from symptomatic tissues were obtained and subcultured. After seven days at 25°C in the dark, the mycelium of a representative culture MFR1 covered the entire plate surface (9 cm diameter). The aerial mycelium of cultures were white and fluffy at first and produced lavender pigment on the back of the cultures in the later stage. After seven days, the cultures produced abundant sickle-shaped macroconidia which have 3 to 5 septations and some oval or oblong microconidia which have 0 to 1 septation. Macroconidia 22.35~46.67 μm (mean 32.11 μm) in length and 4.32~7.72 μm (mean 5.21 μm) in width (n = 100). Microconidia 7.10~21.85 μm (mean 11.62 μm) in length and 2.76~6.84 μm (mean 4.20 μm) in width (n = 100). Based on these characteristics, isolates were tentatively identified as Fusarium sp. (Crous et al. 2021). Pathogenicity was tested on 1-year-old potted seedlings of H. mutabilis by root-zone irrigation inoculation in Sichuan Agricultural University (Jia et al.2019). Conidia suspension (1×107conidia/mL,collected from MFR1 )was poured into the soil along the plant roots. The same amount of distilled water was poured around the roots of the control plants. All inoculated and control plants were incubated in the greenhouse (about 25 ± 2°C). The experiment was performed three times. Within 25 days after inoculation, all plants inoculated with pathogens showed symptoms similar to those in the field, whereas the controls remained symptomless. The pathogen was reisolated from all inoculated plants, and the cultural and morphological characteristics were the same as those of the original isolate. After DNA extraction and PCR amplification, the translation elongation factor 1-alpha (TEF) and RNA polymerase II second largest subunit (RPB2) genes of a representative culture MFR1, were sequenced (O'Donnell et al. 2010) and deposited in GenBank (accession numbers OK334295 and ON316728, respectively). The TEF and RPB2 sequences were 99.7% and 99.39% identical to those of F. oxysporum (MN892354 and MZ198892). The result was confirmed by multilocus phylogenetic analysis. Through morphological identification and molecular analyses, the pathogen was identified as F. oxysporum. F. oxysporum is known to infect cotton (Dowd et al.2004), soybean (Ellis et al.2016) and banana (Fourie et al.2011) among other hosts, but it is the first report of F. oxysporum infecting H. mutabilis in China or worldwide. This disease seriously reduced the survival rate of H. mutabilis and may become an important reason to hinder the increase of H. mutabilis in potted seedlings stage. Moreover, the findings will provide theoretical basis to solve the bottleneck problem affecting the popularization and propagation of H. mutabilis.

Genome skimming as an efficient tool for authenticating commercial products of the pharmaceutically important Paris yunnanensis (Melanthiaceae)

BackgroundParis yunnanensis (Melanthiaceae) is a traditional Chinese medicinal plant of significant pharmaceutical importance. Due to previous taxonomic confusion, a congeneric species, Paris liiana, has been mistaken for P. yunnanensis and cultivated on a large scale, leading to the mixing of commercial products (i.e., seedlings and processed rhizomes) of P. yunnanensis with those of P. liiana. This may have adverse effects on quality control in the standardization of P. yunnanensis productions. As the lack of PCR amplifiable genomic DNA within processed rhizomes is an intractable obstacle to the authentication of P. yunnanensis products using PCR-based diagnostic tools, this study aimed to develop a PCR-free method to authenticate commercial P. yunnanensis products, by applying genome skimming to generate complete plastomes and nrDNA arrays for use as the molecular tags.ResultsBased on a dense intraspecies sampling of P. liiana and P. yunnanensis, the robustness of the proposed authentication systems was evaluated by phylogenetic inferences and experimental authentication of commercial seedling and processed rhizome samples. The results indicate that the genetic criteria of both complete plastomes and nrDNA arrays were consistent with the species boundaries to achieve accurate discrimination of P. yunnanensis and P. liinna. Owing to its desirable accuracy and sensitivity, genome skimming can serve as an effective and sensitive tool for monitoring and controlling the trade of P. yunnanensis products.ConclusionThis study provides a new way to solve the long-standing problem of the molecular authentication of processed plant products due to the lack of PCR amplifiable genomic DNA. The proposed authentication system will support quality control in the standardization of P. yunnanensis products in cultivation and drug production. This study also provides molecular evidence to clarify the long-standing taxonomic confusion regarding the species delimitation of P. yunnanensis, which will contribute to the rational exploration and conservation of the species.

Polymorphisms of mtDNA in the D-loop region moderate the associations of BMI with HOMA-IR and HOMA-β among women with polycystic ovary syndrome: a cross-sectional study.

Polycystic ovary syndrome (PCOS) is one of the leading causes of infertility in women of childbearing age, and many patients with PCOS have obesity and insulin resistance (IR). Although obesity is related to an increased risk of IR, in clinical practice, PCOS patients exhibit different effects on improving insulin sensitivity after weight loss. Therefore, in the present study, we aimed to examine the moderating effect of polymorphisms of mtDNA in the D-loop region on the associations of body mass index (BMI) with the homeostasis model assessment of insulin resistance index (HOMA-IR) and pancreatic β cell function index (HOMA-β) among women with PCOS. Based on a cross-sectional study, women with PCOS were recruited from the Reproductive Center of the First Affiliated Hospital of Anhui Medical University from 2015 to 2018. A total of 520 women who were diagnosed with PCOS based on the revised 2003 Rotterdam criteria were included in the study. Peripheral blood was collected from these patients, followed by DNA extraction, PCR amplification, and sequencing at baseline. HOMA-IR and HOMA-β were calculated according to blood glucose-related indices. Moderating effect models were performed with BMI as an independent variable, polymorphisms of mtDNA in the D-loop region as moderators, and ln (HOMA-IR) and ln (HOMA-β) as dependent variables. To verify the stability of moderating effect, sensitivity analysis was performed withthe quantitative insulin sensitivity check index (QUICKI), fasting plasma glucose/fasting insulin (G/I), and fasting insulin as dependent variables. BMI was positively associated with ln (HOMA-IR) and ln (HOMA-β) (β = 0.090, p < 0.001; β = 0.059, p < 0.001, respectively), and the relationship between BMI and ln (HOMA-IR) or ln (HOMA-β) was moderated by the polymorphisms of mtDNA in the D-loop region. Compared with the respective wild-type, the variant -type of m.16217 T > C enhanced the association between BMI and HOMA-IR, while the variant-type of m.16316 A > G weakened the association. On the other hand, the variant-type of m.16316 A > G and m.16203 A > G weakened the association between BMI and HOMA-β, respectively. The results of QUICKI and fasting insulin as dependent variables were generally consistent with HOMA-IR, and the results of G/I as dependent variables were generally consistent with HOMA-β. Polymorphisms of mtDNA in the D-loop region moderate the associations of BMI with HOMA-IR and HOMA-β among women with PCOS.

Equine blastocyst re-expansion rate, quality, and sex following embryonic collapse using needle perforation of blastocoel and subsequent PCR amplification of blastocoel DNA

Collapse of equine blastocysts following needle perforation of the blastocoel prior to vitrification (Ferris et al., Journal of Equine Veterinary Science. 2016; 100:64-65) and collection and amplification of the free DNA blastocoel fluid may be used for sexing embryos (Herrera et al., Theriogenology. 2015; 83:415-420). The present study aimed to evaluate equine blastocyst re-expansion rate, quality, and sex following manual perforation of the blastocoel and PCR amplification of blastocoel fluid DNA. Embryos (n=19) were collected on day 8 after ovulation, assessed initially and transferred to a 50µL droplet of holding medium in which the blastocoel was manually perforated with a 26g hypodermic needle. Within one minute of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at -20°C until PCR. Some of the collapsed embryos (n=11) were moved and immersed in 2mL of holding medium and cultured in isothermal boxes at 25°C. After 24h, embryo size and quality were reassessed. Post-culture, collapsed blastocysts had partially re-expanded to their initial diameter with a mean expansion rate of 68.8%. Quality of re-expanded embryos was Grade 2 (good, 6/11), Grade 3 (poor, 4/11), and Grade 4 (degenerated, 1/11) (McKinnon,Squires, J. Am. Vet Med. Assoc. 1988; 192:401-406). Non-perforated embryos (n=10) used as controls were initially Grade 1 (excellent, 8/10) and Grade 2 (2/10) and, after 24h of culture, Grade 1 (9/10) and Grade 2 (1/10) with a 25.9% growth rate. Blastocoel fluid from all collapsed embryos (n=19) was subjected to double PCR amplification of the male TSPY gene with blood of an adult male and female horse used as controls. Gene amplification indicated 57.9% (11/19) of embryos were male and non-amplification indicated 31.6% (6/19) of embryos were female. Gene amplification was inconclusive for 10.5% (2/19) of the embryos even though they were completely collapsed. Thus, needle-induced leakage and collapse of the blastocoel resulted in a high rate of blastocysts re-expansion (69%) with many embryos (54%) achieving good quality and potential for transfer as either male or female embryos. In summary, while the number of potentially transferable embryos that recovered from collapse was less than in the control group, the success of these preliminary results warrants further development to decrease damage and increase the culture re-expansion rate and quality of transferable male or female embryos for field application in an equine embryo transfer program.

Direct Nanopore Sequencing of Human Cytomegalovirus Genomes from High-Viral-Load Clinical Samples.

Nanopore sequencing is becoming increasingly commonplace in clinical settings, particularly for diagnostic assessments and outbreak investigations, due to its portability, low cost, and ability to operate in near real-time. Although high sequencing error rates initially hampered the wider implementation of this technology, improvements have been made continually with each iteration of the sequencing hardware and base-calling software. Here, we assess the feasibility of using nanopore sequencing to determine the complete genomes of human cytomegalovirus (HCMV) in high-viral-load clinical samples without viral DNA enrichment, PCR amplification, or prior knowledge of the sequences. We utilised a hybrid bioinformatic approach that involved assembling the reads de novo, improving the consensus sequence by aligning reads to the best-matching genome from a collated set of published sequences, and polishing the improved consensus sequence. The final genomes from a urine sample and a lung sample, the former with an HCMV to human DNA load approximately 50 times greater than the latter, achieved 99.97 and 99.93% identity, respectively, to the benchmark genomes obtained independently by Illumina sequencing. Thus, we demonstrated that nanopore sequencing is capable of determining HCMV genomes directly from high-viral-load clinical samples with a high accuracy.

Type-specific quantification of particulate methane monooxygenase gene of methane-oxidizing bacteria at the oxic-anoxic interface of a surface paddy soil by digital PCR.

Aerobic methane-oxidizing bacteria (MOB) play an important role in mitigating methane emissions from paddy fields. In this study, we developed a differential quantification method for the copy number of pmoA genes of type Ia, Ib, and IIa MOB in paddy field soil using chip-based digital PCR. Three probes specific to the pmoA of type Ia, Ib, and IIa MOB worked well in digital PCR quantification when genomic DNA of MOB isolates and PCR-amplified DNA fragments of pmoA were examined as templates. When pmoA genes in the surface soil layer of a flooded paddy were quantified by digital PCR, the copy numbers of type Ia, Ib, and IIa MOB were 105 -106 , 105 -106 , and 107 copies g-1 dry soil, respectively, with the highest values in the top 0-2-mm soil layer. Especially, the copy numbers of type Ia and Ib MOB increased by 240% and 380% at the top layer after soil flooding, suggesting that the soil circumstances at the oxic-anoxic interfaces were more preferential for growth of type I MOB than type II MOB. Thus, type I MOB likely play an important role in the methane consumption at the surface paddy soil.

On the causes, consequences, and avoidance of PCR duplicates: Towards a theory of library complexity.

Library preparation protocols for most sequencing technologies involve PCR amplification of the template DNA, which open the possibility that a given template DNA molecule is sequenced multiple times. Reads arising from this phenomenon, known as PCR duplicates, inflate the cost of sequencing and can jeopardize the reliability of affected experiments. Despite the pervasiveness of this artefact, our understanding of its causes and of its impact on downstream statistical analyses remains essentially empirical. Here, we develop a general quantitative model of amplification distortions in sequencing data sets, which we leverage to investigate the factors controlling the occurrence of PCR duplicates. We show that the PCR duplicate rate is determined primarily by the ratio between library complexity and sequencing depth, and that amplification noise (including in its dependence on the number of PCR cycles) only plays a secondary role for this artefact. We confirm our predictions using new and published RAD-seq libraries and provide a method to estimate library complexity and amplification noise in any data set containing PCR duplicates. We discuss how amplification-related artefacts impact downstream analyses, and in particular genotyping accuracy. The proposed framework unites the numerous observations made on PCR duplicates and will be useful to experimenters of all sequencing technologies where DNA availability is a concern.

Rapid Detection and Identification of Fasciola spp. and Dicrocoelium spp. Isolated from the Ruminant Livestock of Northwest Iran Using High-Resolution Melting Analysis (HRM).

The liver flukes of the Fasciola species and Dicrocoelium spp. are recognised as parasites of domestic and wild herbivores. Both species of F. hepatica and F. gigantica as well as D. dendriticum are distributed in Iran. The present study aimed to identify Fasciola spp. and Dicrocoelium spp. using mitochondrial Cox1 (cytochrome c oxidase I) gene by HRM method. Totally, thirty infected liver specimens were collected from the sheep (n:23) and cattle (n:7) at the abattoirs of Qazvin Province, northwest Iran in 2022. DNA extraction and PCR amplification of Cox1 gene were conducted by HRM technique. DnaSP v.5.0 was used for compression of diversity indices of ribosomal 28S rDNA and mitochondrial Cox1 markers of Dicrocoelium spp. The taxonomic status of Dicrocoelium spp. was performed by sequencing and phylogenetic analysis. Overall, 26 and 4 isolates were identified as F. hepatica and F. gigantica, respectively. D. dendriticum was the sole infecting species of Dicrocoelium revealed by HRM analysis. Genomic analysis showed a moderate (28S rDNA genes: 0.600±0.215) to high (Cox1: 0.733±0.155) haplotype diversity for D. dendriticum. The parasite-dependent mitochondrial gene (Cox1) could identify a higher genetic diversity of D. dendriticum compared to nuclear 28S rDNA gene. HRM technique in the present study found to be a reliable technique for identification and genetic diversity of liver flukes but more comprehensive and in-depth studies in different parts of the country are needed.