DNA PCR Research Articles

One-instrument, objective microsatellite instability analysis using high-resolution melt.

In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers. The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type. The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status. The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance. Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors. Novel technologies have been applied and concepts such as tumor mutation burden have been introduced. However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator. In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue. The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles. The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study. The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.

Validation and forensic application of a new 36 X-STR loci multiplex system

Abstract X-chromosomal short tandem repeats (X-STRs) are indispensable in specific cases, distinct from autosomal and Y chromosome genetic markers. SureID® X37 is an innovative six-color fluorescence multiplex detection system that can simultaneously amplify 36 X-STR loci (including DXS6795, DXS7132, DXS8378, DXS10101, DXS10103, DXS10079, DXS10134, GATA172D05, HPRTB, DXS6810, DXS10135, DXS6797, DXS10074, DXS7424, DXS9902, DXS7423, DXS10148, DXS10162, DXS6809, GATA31E08, DXS6803, DXS10075, DXS6807, DXS10164, DXS6789, DXS10146, DXS981, DXS9895, DXS101, DXS6800 DXS907, and DXS8377) and the Amelogenin locus. In this study, we validated its suitability for forensic identification per SWGDAM (Scientific Working Group on DNA analysis methods) guidelines, including PCR conditions, precision, accuracy, case-type samples, sensitivity, repeatability, reproducibility, species specificity, stability, stutter calculation, and DNA mixtures. Additionally, 577 Han Chinese individuals were used to investigate the utility of the system in forensic population genetics. The results indicated that the system is sensitive, stable, and reliable, and is suitable for typical forensic case. Subsequent population investigations confirmed that it serves as a potent supplementary tool in forensic applications.

The phenotypic and genotypic antibiotic susceptibility of vaginal Lactobacillus with potential probiotic properties isolated from healthy women in northern Iran

Background and Objectives: This study assesses the antibiotic susceptibility of vaginal Lactobacillus strains and provides data for determining the prevalence of certain antibiotic resistance genes in the new strains of lactobacilli serving as probiot- ics and selected from healthy women in northern Iran. Materials and Methods: One hundred premenopausal non-pregnant women in the reproductive age range of 22-50 years participated in this study. The potential probiotic vaginal lactobacilli used in the study included Lactobacillus crispatus (34.2%), Lactobacillus gasseri (26.3%), Lactobacillus johnsonii (10.5%), Lactobacillus acidophilus (15.7%) and Lactoba- cillus jensenii (13.1%). The phenotypic antibiotic susceptibility of the strains was determined by E test and DNA extraction and PCR were performed to examine the antibiotic resistance genes. Results: 38 potential probiotic vaginal lactobacilli were isolated. All the strains of lactobacilli were resistant to metronida- zole and trimethoprim/sulfamethoxazole and all of the strains were susceptible to ampicillin and chloramphenicol antibiotics. The results showed that ermB, ermC, and ermA genes were observed in the strains of Lactobacillus acidophilus. Metronida- zole resistance (nim) gene was also found in one strain of Lactobacillus crispatus and Lactobacillus johnsonii. The amino- glycoside resistance (aac6'-aph2") gene was observed in 8% of the strains. Also, tetM, tetK and tetW genes were found in more than 80% of the Lactobacillus strains. Conclusion: The antimicrobial susceptibility of vaginal lactobacilli is an important criterion for establishing whether or not the organism is a probiotic. A high level of resistance to clinical antibiotics, such as metronidazole and aminoglycosides, was demonstrated. Antibiotic resistant genes also appeared widely in vaginal lactobacilli.

Comparison of DNA quantity and quality from fecal samples of mammals transported in ethanol or lysis buffer

Using fecal microbial community profiles through sequencing approaches helps to unravel the intimate interplay between health, wellness, and diet in wild animals with their environment. Ensuring the proper preservation of fecal samples before processing is crucial to ensure reliable results. In this study, we evaluated the efficiency of two different preservation methods, considering the following criteria: DNA yield, quality and integrity, and microbial community structure based on Oxford Nanopore amplicon sequencing of the V3-V4 region of bacterial 16S rRNA and protozoa 18S rRNA genes. Eighteen matched pairs of mammalian fecal samples were collected and transported in 99.8% ethanol and lysis buffer; processing occurred between 55 and 461 days post-collection. Wilcoxon signed-rank tests were used to analyze quantitative measurements for paired samples. The A260/280 ratio, a measure of nucleic acid purity, was assessed descriptively for each media, and the Bartlett test evaluated dispersion of this ratio. A Fisher test was performed to compare the number of positive reactions for DNA extraction or PCR amplification of the 16S and 18S rRNA genes between both media. The concentration of total DNA and amplicons, as well as the number of reads obtained in sequencing, was significantly higher in the samples preserved with lysis buffer compared to ethanol, with magnitudes up to three times higher. Electrophoretic analysis of total DNA and amplicons further confirmed superior DNA integrity in lysis buffer preserved samples. The A260/280 values obtained using the lysis buffer were of optimal purity (mean: 1.92) and with little dispersion (SD: 0.27); on the other hand, the ethanol samples also presented an excellent average quality (mean: 1.94), but they were dispersed (SD: 1.10). For molecular studies using mammalian feces, the lysis buffer reagent proved to be a reliable solution for their collection, conservation, and storage.

Dynamics and prognostic value of plasma cell-free DNA PCR in patients with invasive aspergillosis and mucormycosis.

We show that Aspergillus and Mucorales plasma cell-free DNA PCR can be used not only to noninvasively diagnose patients with invasive fungal disease but also to correlate the baseline cycle threshold with survival outcomes, thus potentially allowing the identification of patients at risk for poor outcomes, who may benefit from more targeted therapies.

P16 overexpression identifies oncogenic high-risk HPV infection in non-oropharyngeal squamous cell carcinoma of the head and neck.

Human papillomavirus (HPV) is an increasing risk factor for cancer. HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) is associated with a favorable outcome. Blockstaining for p16 is a surrogate marker for HPV+ OPSCC. In oral and laryngeal squamous cell carcinoma (OSCC/LSCC), the relevance of p16 immunohistochemistry, alone or in combination with other cell cycle-related proteins, to identify HPV-driven non-OPSCC is less well understood. We stained for p16, pRb, cyclin D1, and p53 in 327 HNSCC. In 310 OPSCC, HPV-status was assessed by HPV DNA PCR. In 119 non-OPSCC, RNA in situ hybridization was additionally performed. HPV-status was correlated with staining patterns, p53 and clinical data. The OPSCC showed blockstaining for p16 in 36%, 8% were equivocal. Of these, HPV-testing was performed in 57%, and 53% were positive for HPV DNA. HPV-association correlated with absence of pRb and cyclin D1 and favorable outcome. In non-OPSCC, 18% showed p16-blockstaining, and 13% showed E6/E7 RNA. Six of seven HPV+ OSCC and 8/8 LSCC lost pRb and cyclin D1. Compared to HPV-negative counterparts, patients with HPV+ cancers had lower rates of alcohol consumption and keratinizing morphology. HPV-positive OSCC had a longer overall survival (p < 0.05). HPV subtype 16 was the most common. We conclude that HPV-positive non-OPSCC are associated with p16 overexpression and low levels of pRb and cyclin D1. High expression of pRb and cyclin D1 indicates HPV-negativity.

A survey of gastrointestinal helminth infestation in smallholder backyard pigs and the first molecular identification of the two zoonotic helminths Ascaris suum and Trichuris suis in Myanmar

BackgroundParasitic infestations have a substantial economic impact on pig production. This study aimed to investigate the gastrointestinal (GI) helminths in pigs and to molecularly characterise two important nematodes, Ascaris and Trichuris species.Materials and methodsA total of 500 pig faecal samples were collected from small holder backyard pig farms in five townships within Nay Pyi Taw, Myanmar. Microscopic examination was conducted to estimate the prevalence of GI helminth infestation in the pigs. DNA extraction and PCR were performed on faecal samples that were morphologically positive for Ascaris and Trichuris eggs. Molecular analysis was then conducted to characterise A. suum and T. suis, the most common and zoonotic helminths.ResultsAccording to microscopic examination, 69.2% (346/500) were positive for GI helminth eggs. The GI helminth species observed were A. suum, Strongyle, Strongyloides spp., T. suis, Metastrongylus spp., Hyostrongylus spp., Fasciolopsis spp., Paragonimus spp., and Schistosoma spp., with occurrences of 34.8%, 29.6%, 21.4%, 20.0%, 4.0%, 1.6%, 1.0%, 1.0%, and 0.4%, respectively. Mixed infections of GI helminths were noted in 31.0% of the samples. Overall, sampled pigs excreted mostly low levels (< 100 EPG) or moderate levels (> 100–500 EPG) of GI helminth eggs. The highest mean EPG for each parasite species was noted in A. suum. The presence of A. suum and T. suis was confirmed molecularly. The sequences of the internal transcribed spacer 1 (ITS1) region of A. suum showed high similarity with previously reported sequences. Likewise, the sequences of T. suis exhibited high similarity with the sequences reported from humans and pigs. Age was noted as an associated factor (P < 0.05) for GI helminth infection status.ConclusionsIn this report, A. suum and T. suis were molecularly identified for the first time in Myanmar. It is important to extend the information among the farmers to be aware of the necessity of preventing zoonotic parasites by practicing regular deworming, proper use of anthelmintics and maintaining hygienic conditions in their pig farms.

A Basic Guide to the Propagation and Manipulation of the Clubroot Pathogen, Plasmodiophora brassicae.

Clubroot caused by the obligate parasite Plasmodiophora brassicae is a devastating disease affecting the canola industry worldwide. The socio-economic impact of clubroot can be significant, particularly in regions where Brassica crops are a major agricultural commodity. The disease can cause significant crop losses, leading to reduced yield and income for farmers. Extensive studies have been conducted to understand the biology and genetics of the pathogens and develop more effective management strategies. However, the basic procedures used for pathogen storage and virulence analysis have not been assembled or discussed in detail. As a result, there are discrepancies among the different protocols used today. The aim of this article is to provide a comprehensive and easily accessible resource for researchers who are interested in replicating or building upon the methods used in the study of the clubroot pathogen. Here, we discuss in detail the methods used for P. brassicae spore isolation, inoculation, quantification, propagation, and molecular techniques such as DNA extraction and PCR. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extraction of Plasmodiophora brassicae resting spores and propagation Support Protocol 1: Evans blue staining to identify resting spore viability Support Protocol 2: Storage of Plasmodiophora brassicae Basic Protocol 2: Generation of single spore isolates from P. brassicae field isolates Basic Protocol 3: Phenotyping of Plasmodiophora brassicae isolates Basic Protocol 4: Genomic DNA extraction from Plasmodiophora brassicae resting spores Basic Protocol 5: Molecular detection of Plasmodiophora brassicae.

A method for noninvasive individual genotyping of black-footed cat (Felis nigripes).

The black-footed cat (Felis nigripes) is endemic to the arid regions of southern Africa. One of the world's smallest wild felids, the species occurs at low densities and is secretive and elusive, which makes ecological studies difficult. Genetic data could provide key information such as estimates on population size, sex ratios, and genetic diversity. In this study, we test if microsatellite loci can be successfully amplified from scat samples that could be noninvasively collected from the field. Using 21 blood and scat samples collected from the same individuals, we statistically tested whether nine microsatellites previously designed for use in domestic cats can be used to identify individual black-footed cats. Genotypes recovered from blood and scat samples were compared to assess loss of heterozygosity, allele dropout, and false alleles resulting from DNA degradation or PCR inhibitors present in scat samples. The microsatellite markers were also used to identify individuals from scats collected in the field that were not linked to any blood samples. All nine microsatellites used in this study were amplified successfully and were polymorphic. Microsatellite loci were found to have sufficient discriminatory power to distinguish individuals and identify clones. In conclusion, these molecular markers can be used to monitor populations of wild black-footed cats noninvasively. The genetic data will be able to contribute important information that may be used to guide future conservation initiatives.

Significant beneficial effects of 12-weeks add-on yoga therapy on antipsychotic-stabilized schizophrenia patients through epigenetic modulation: novel findings from a randomized controlled study

IntroductionComplementary and alternative therapy, especially yoga, is emerging as an important treatment modality for various complex disorders. Yoga therapy has reportedly been demonstrated to exhibit clinical benefits in schizophrenia. However, the modulatory effects of yoga therapy on the pathobiological pathways of schizophrenia are inadequately explored. Immune dysregulation is a widely recognized etiopathological construct of schizophrenia. It is not precisely known whether yoga therapy can modulate the expression of immune molecules by regulating gene expression and epigenetic processes in schizophrenia.ObjectivesTo understand the impact of 12-weeks add-on yoga therapy on the immune-inflammatory pathway in schizophrenia by examining plasma levels and gene expression levels of cytokines and complement proteins as well as by profiling promoter DNA methylation pattern of genes coding for cytokines and complement proteins.MethodsFifty-seven schizophrenia patients fulfilling DSM-V criteria were recruited into the study and randomized into Yoga therapy (n=28) and waitlist control (n=29) groups. Plasma levels of IL-1β, IL-6, IL-10, IL-17, C1q, C2, C3, C4, C5, C5a, Factor B and Factor H by Multiplex Suspension Assay, quantification of gene expression of Il1b, Il6, Il10, IL17, C3, C4 and C5 genes by quantitative PCR and promoter DNA methylation of Il1b, Il6, Il10, Il17, C3, C4 and C5 genes by pyrosequencing were carried out in all the study participants.ResultsPlasma levels of IL-1β (Z score= 2.42, p=0.02) dropped significantly and C2 (Z score= 2.24, p=0.03) levels increased after 12-weeks of yoga therapy. The expression of Il1b (Z score=2.45, p=0.01) and Il6 (Z score=2.07, p=0.04) genes were significantly downregulated, while the levels of C4 (Z score=2.23, p=0.03) gene was upregulated in schizophrenia patients of yoga therapy group. Two CpG sites in the promoter region of Il1b (all p≤0.05) and Il6 (all p≤0.05) genes and three CpG sites in the promoter region of C4 (all p&lt;0.05) gene were hypermethylated, while two CpG sites in the gene body of Il6 (all p≤0.05) gene and two CpG sites in the promoter region of Il10 (all p ≤0.05) gene were hypomethylated after 12-weeks of yoga therapy in schizophrenia patients.ConclusionsOur findings provide important insights into the mode of action of yoga therapy in schizophrenia. This study for the first time reports the epigenetic effects of yoga therapy on immune-inflammatory pathway in schizophrenia.Disclosure of InterestNone Declared

A Tomato-Free Period Delays and Reduces Begomovirus Disease in Processing Tomato Fields in a Complex Agroecosystem in Central Brazil.

A mandatory tomato-free period (TFP) was implemented in the state of Goiás, Brazil, in 2007 to help manage diseases caused by whitefly-transmitted begomoviruses. The impact of the TFP was examined in five locations across three states in Central Brazil from 2013 to 2016. Surveys revealed significant differences in begomovirus disease incidence among locations, i.e., low in Guaíra-TFP and Patos de Minas-TFP; moderate-high in Itaberaí-TFP and Morrinhos-TFP; and high in the non-TFP (NTFP) control, Cristalina-NTFP. PCR tests and DNA sequencing were used to validate the symptoms and showed that all collected symptomatic plant samples were infected with tomato severe rugose virus (ToSRV), a common indigenous bipartite begomovirus. Early season surveys (20 to 40 days after transplants [DAT]) in Itaberaí-TFP and Morrinhos-TFP revealed significantly less begomovirus disease in fields established sooner after the TFP (0 to 2 months) compared with incidences in (i) equivalent early planted fields in the Cristalina-NTFP control and (ii) fields established longer after the end of the TFP (>2 to 5 months). Whitefly infestation of crops was detected year-round in all locations and years, and all tested adults were classified in the Bemisia tabaci MEAM1 cryptic species. Infestation levels were significantly higher during the summer but did not vary significantly among locations. Results of monthly monitoring of adult whiteflies for general begomovirus and ToSRV were positively correlated and were indicators of disease incidence in the field. Notably, ToSRV was not detected in whiteflies collected from nontomato plants during the TFP, and there was a longer lag period before detection in whiteflies collected from processing tomatoes for Itaberaí-TFP and Morrinhos-TFP compared with Cristalina-NTFP. Taken together with the low levels of ToSRV infection detected in potential nontomato reservoir hosts at all locations, our results revealed low levels of primary inoculum during the TFP. Thus, even in a complex agroecosystem with year-round whitefly infestation of crops, the TFP was beneficial due to delayed and reduced begomovirus disease pressure during a critical stage of plant development (first month) and for favoring low levels of primary inoculum. Thus, we concluded that the TFP should be part of a regional integrated pest management (IPM) program targeting ToSRV in Brazil.

Validation of the VisionArray® Chip Assay for HPV DNA Testing in Histology Specimens of Oropharyngeal Squamous Cell Carcinoma

BackgroundThe detection of human papillomavirus (HPV) has several implications in the diagnostic work-up and management of oropharyngeal squamous cell carcinoma (OPSCC). The choice of HPV detection assay and testing algorithms differ across institutions and vary in cost, detection targets, technical feasibility, and turnaround time. In this study, we aimed to validate the VisionArray® HPV Chip for formalin-fixed and paraffin-embedded (FFPE) samples of OPSCC using the previously applied standard pan-HPV DNA PCR assay as a reference.MethodsThe validation cohort consisted of FFPE tissue samples from patients previously diagnosed with HPV DNA-positive OPSCC (n = 80), HPV DNA-negative OPSCC (n = 21), and a benign group of tumor samples consisting of Warthin’s tumors (n = 20) and branchial cleft cysts of the lateral neck (n = 14). All samples were tested with p16 immunohistochemistry, pan-HPV DNA PCR, and the VisionArray® HPV Chip.ResultsThe overall sensitivity and specificity of the VisionArray® HPV Chip assay were 100% [95% CI 95.5%; 100.0%] and 96.3% [95% CI 87.3%; 99.6%] and the positive predictive value and negative predictive value were 97.6% [95% CI 91.5%; 99.7%] and 100% [95% CI 93.2%; 100%], respectively.ConclusionsThe VisionArray® HPV Chip assay can be recommended for high-risk HPV testing in FFPE tissue samples from OPSCC, providing both a fast and simultaneous genotyping for 41 clinically relevant HPV types.

Investigation on disease incidence, distribution and diversity of Begomoviruses infecting cucurbitaceous crops in North Bengal, India

Aim: To study the disease incidence and diversity of Begomovirus infection and crop loss due to spread of virus across different districts of North Bengal. Methodology: Several fields in different districts of North Bengal were surveyed and potential begomoviral-infected samples were collected for further analysis. Total DNA extraction, PCR analysis, sequencing and phylogenetic analysis were conducted. Results: During 2019-2021, ten fields of each district were surveyed and begomoviral occurrence was observed in almost every field. The disease incidence in different districts varied from 15-45%. The highest disease (42.5%) incidence of was observed in the Darjeeling district while the lowest (15%) was recorded from Alipurduar district. The maximum crop loss due to Begomovirus was 64-100% in bitter gourd, 40-80% in cucumber and 20-30% in wax gourd. 153 samples from 6 different cucurbits were collected and analysed. PCR using AV494/AC1048 primers that amplified coat protein region (~550bp) showed 74% of the samples as positive. Upon sequencing and BLAST analysis, ToLCNDV, SLCCV and BGYVV were detected. In phylogenetic analysis, the isolates clustered in three separate clades based on Begomovirus species irrespective of the hosts. Present isolates showed more closeness to Indian and Bangladesh isolates. Interpretation: The documentation of disease severity caused by Begomovirus is of extreme importance for food security of the people. Present study revealed that Begomovirus is present in all the crop field studied. The results indicated an urgent need for the management of the viruses to save crops. Key words: Begomovirus, Cucurbitaceous crops, Disease incidence, North Bengal

Reappraisal of p16 for Determining HPV Status of Head and Neck Carcinomas Arising in HPV Hotspots.

In an era of head and neck oncology where HPV status will soon dictate patient management, reliable HPV detection is critical. P16 immunohistochemistry (IHC) is currently recommended as the test of choice for oropharyngeal squamous cell carcinomas (OPSCCs). The purpose of this study was to determine the performance characteristics of p16 IHC based on a large clinical experience of squamous cell carcinomas (SCC) arising from HPV hot-spot regions of the head and neck. Consecutive OPSCCs, sinonasal SCCs, and metastatic SCCs of unknown primary sites were evaluated for the presence of HPV by p16 IHC and PCR-based HPV DNA testing as part of clinical care. For discrepant cases, high-risk HPV E6/E7 mRNA in situ hybridization (ISH) and, when possible, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MassArray) genotyping were performed. 746 cancers underwent HPV testing by p16 IHC and DNA PCR genotyping. There was a 95.6% concordance between the 2 assays. Of the 33 discrepant cases, 32 cases (4.3%) were p16 positive but HPV DNA negative. In these cases, 68% were positive for mRNA ISH, invariably related to a non-16 HPV genotype. P16 IHC had an overall accuracy of 98.8%, a sensitivity of 99.8%, and a specificity of 92.1%. P16 IHC is a sensitive and specific assay for determining HPV status. HPV DNA PCR appears vulnerable to HPV genotype diversity and is prone to missing rare non-16 genotypes. HPV mRNA ISH is a practical and reliable direct measure of HPV that may help eliminate the small number of false-positive p16 cases and avoid potential patient harm related to erroneous HPV classification.

Sequencing vs. amplification for the estimation of allele dosages in sugarcane (Saccharum spp.).

Detecting single-nucleotide polymorphisms (SNPs) in a cost-effective way is fundamental in any plant breeding pipeline. Here, we compare three genotyping techniques for their ability to reproduce the allele dosage of SNPs of interest in sugarcane (Saccharum spp.). To identify a reproducible technique to estimate allele dosage for the validation of SNP markers, the correlation between Flex-Seq, kompetitive allele-specific PCR (KASP), and genotyping-by-sequencing and restriction site-associated DNA sequencing (GBS+RADseq) was determined for a set of 76 SNPs. To find alternative methodologies for allele dosage estimation, the KASP and Flex-Seq techniques were compared for the same set of SNPs. For the three techniques, a population of 53 genotypes from the diverse sugarcane panel of the Centro de Investigación de la Caña de Azúcar (Cenicaña), Colombia, was selected. The average Pearson correlation coefficients between GBS+RADseq and Flex-Seq, GBS+RADseq and KASP, and Flex-Seq and KASP were 0.62 ± 0.27, 0.38 ± 0.27, and 0.38 ± 0.30, respectively. Flex-Seq reproduced the allele dosages determined using GBS+RADseq with good levels of precision because of its depth of sequencing and ability to target specific positions in the genome. Additionally, Flex-Seq outperformed KASP by allowing the conversion of a higher number of SNPs and a more accurate estimation of the allele dosage. Flex-Seq has therefore become the genotyping methodology of choice for marker validation at Cenicaña.

Using Chromatin Immunoprecipitation (ChIP) to Study the Chromatin State in Drosophila.

The chromatin state plays an important role in regulating gene expression, which affects organismal development and plasticity. Proteins, including transcription factors, chromatin modulatory proteins, and histone proteins, usually with modifications, interact with gene loci involved in cellular differentiation, function, and modulation. One molecular method used to characterize protein-DNA interactions is chromatin immunoprecipitation (ChIP). ChIP uses antibodies to immunoprecipitate specific proteins cross-linked to DNA fragments. This approach, in combination with quantitative PCR (qPCR) or high-throughput DNA sequencing, can determine the enrichment of a certain protein or histone modification around specific gene loci or across the whole genome. ChIP has been used in Drosophila to characterize the binding pattern of transcription factors and to elucidate the roles of regulatory proteins in gene expression during development and in response to environment stimuli. This review outlines ChIP procedures using tissues from the Drosophila nervous system as an example and discusses all steps and the necessary optimization.

Factors Underlying the Difference in Response to Fecal Microbiota Transplantation Between IBS Patients with Severe and Moderate Symptoms

BackgroundPrevious studies showed that patients with Severe IBS respond better to fecal microbiota transplantation (FMT) than do those with Moderate IBS.AimsThe present study aimed to determine the effects of the transplant dose, route of administering it and repeating FMT on this difference.MethodsThis study included 186 patients with IBS randomized 1:1:1 into groups with a 90-g transplant administered once to the colon (LI), once to the duodenum (SI), or twice to the distal duodenum twice (repeated SI). The patients provided a fecal sample and were asked to complete three questionnaires at baseline and at 3, 6, and 12 months after FMT. The fecal bacteria composition and Dysbiosis index were analyzed using 16 S rRNA gene PCR DNA amplification/probe hybridization covering regions V3–V9.ResultsThere was no difference in the response rates between severe IBS and moderate IBS for SI and repeated SI at all observation intervals after FMT. In the LI group, the response rate at 3 months after FMT was higher for moderate IBS than for severe IBS. The levels of Dorea spp. were higher and those of Streptococcus salivarius subsp. Thermophilus, Alistipes spp., Bacteroides and Prevotella spp., Parabacteroides johnsoni and Parabacteroides spp. were lower in moderate IBS than in severe IBS.ConclusionsThere was no difference in the response to FMT between severe and moderate IBS when a 90-g transplant was administered to the small intestine. The difference in the bacterial profile between severe and moderate IBS may explain the difference in symptoms between these patients. (www.clinicaltrials.gov: NCT04236843).

Improved DNA Extraction and Amplification Strategy for 16S rRNA Gene Amplicon-Based Microbiome Studies.

Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent ('Rapid' protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel 'Rapid' method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the 'Rapid' protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel 'Rapid' DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel 'Rapid' DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing.

Developmental validation of the STRSeqTyper122 kit for massively parallel sequencing of forensic STRs.

Massively parallel sequencing allows for integrated genotyping of different types of forensic markers, which reduces DNA consumption, simplifies experimental processes, and provides additional sequence-based genetic information. The STRseqTyper122 kit genotypes 63 autosomal STRs, 16 X-STRs, 42 Y-STRs, and the Amelogenin locus. Amplicon sizes of 117 loci were below 300bp. In this study, MiSeq FGx sequencing metrics for STRseqTyper122 were presented. The genotyping accuracy of this kit was examined by comparing to certified genotypes of NIST standard reference materials and results from five capillary electrophoresis-based kits. The sensitivity of STRseqTyper122 reached 125pg, and > 80% of the loci were correctly called with 62.5pg and 31.25pg input genomic DNA. Repeatability, species specificity, and tolerance for DNA degradation and PCR inhibitors of this kit were also evaluated. STRseqTyper122 demonstrated reliable performance with routine case-work samples and provided a powerful tool for forensic applications.

A direct PCR approach with low-biomass insert opens new horizons for molecular sciences on cryptogam communities.

Molecular sequence data have transformed research on cryptogams (e.g., lichens, microalgae, fungi, and symbionts thereof) but methods are still strongly hampered by the small size and intermingled growth of the target organisms, poor cultivability and detrimental effects of their secondary metabolites. Here, we aim to showcase examples on which a modified direct PCR approach for diverse aspects of molecular work on environmental samples concerning biocrusts, biofilms, and cryptogams gives new options for the research community. Unlike traditional approaches, this methodology only requires biomass equivalent to colonies and fragments of 0.2 mm in diameter, which can be picked directly from the environmental sample, and includes a quick DNA lysis followed by a standardized PCR cycle that allows co-cycling of various organisms/target regions in the same run. We demonstrate that this modified method can (i) amplify the most widely used taxonomic gene regions and those used for applied and environmental sciences from single colonies and filaments of free-living cyanobacteria, bryophytes, fungi, and lichens, including their mycobionts, chlorobionts, and cyanobionts from both isolates and in situ material during co-cycling; (ii) act as a tool to confirm that the dominant lichen photobiont was isolated from the original sample; and (iii) optionally remove inhibitory secondary lichen substances. Our results represent examples which highlight the method's potential for future applications covering mycology, phycology, biocrusts, and lichenology, in particular.IMPORTANCECyanobacteria, green algae, lichens, and other cryptogams play crucial roles in complex microbial systems such as biological soil crusts of arid biomes or biofilms in caves. Molecular investigations on environmental samples or isolates of these microorganisms are often hampered by their dense aggregation, small size, or metabolism products which complicate DNA extraction and subsequent PCRs. Our work presents various examples of how a direct DNA extraction and PCR method relying on low biomass inserts can overcome these common problems and discusses additional applications of the workflow including adaptations.