Gene Sequencing Assays Research Articles

Effect of sodium diacetate on fermentation, aerobic stability, and microbial diversity of alfalfa silage.

Alfalfa (Medicago sativa L.) is a vital source of forage protein for ruminants, yet its ensiling poses challenges due to high buffering capacity and low water-soluble carbohydrates (WSC). This study investigated the impact of sodium diacetate (SDA) on alfalfa silage quality and aerobic stability. SDA was applied at four different rates to wilted alfalfa on a fresh basis: 0g/kg, 3g/kg, 5g/kg, and 7g/kg, and silages were ensiled in laboratory-scale silos for 45days, followed by 7days of aerobic exposure. A 16S rRNA gene sequencing assay using GenomeLab™ GeXP was performed to determine the relationship between dominant isolated lactic acid bacteria species and fermentation characteristics and aerobic stability on silage. The results showed that Lentilolactobacillus brevis, Pediococcus pentosaceus and Enterococcus faecium were the most prevalent bacteria when silos were opened, whereas Weissella paramesenteroides, Bacillus cereus, B. megaterium and Bacillus spp. were most prevalent bacteria after 7days of aerobic exposure. Dry matter, pH, and WSC content were not affected by SDA, but doses above 5g/kg induced a homofermentative process, which increased lactic acid concentration and lactic acid to acetic acid ratio, decreased yeast count during aerobic exposure, and improved aerobic stability. These findings offer useful information for optimizing SDA usage in silage, assuring improved quality and longer storage, and thereby improving animal husbandry and sustainable feed practices.

Utility evaluation of two molecular methods for Leptospira spp. typing in human serum samples

Most of the available genotyping methods were applied and evaluated in Leptospira isolates and only few of them in a relevant sample size of blood specimens but not of sera. The objective of this study was to evaluate the utility of one partial 16S rRNA gene sequencing assay (16S rRNA) and an optimized. Multilocus sequence typing scheme (MLST) for Leptospira typing directly in serum samples. Confirmed leptospirosis patients (n = 228) from Argentina (2005–2016) were randomly included. Septicemic-phase serum samples (n = 228) were studied by two genotyping methods. Available immune-phase serum samples of the included patients (n = 159) were studied by MAT to compare serological and molecular results. In culture-proven cases (n = 8), genotyping results between clinical samples and isolates were compared. Typing success rate (TSR) was 21.9% for 16S rRNA and 11.4% for MLST (full allelic profile) and a positive trend in both TSR during the study period was observed. Two species (L. interrogans and L. borgpertesenii) were identified by both methods and MLST assigned 8 different STs. The probable serogroups identified by MLST were coincident with the presumptive infecting serogroups identified by MAT, but with different frequencies. The three serogroups (Canicola, Sejroe and Icterohaemorrhagiae) most frequently identified by MAT were also genotyped by MLST. Typing results via 16S rRNA and MLST in clinical samples and isolates of culture-proven cases, were consistent except for one case. Performance of partial 16S rRNA gene sequencing assay and the optimized MLST scheme directly in sera may increase and improve the knowledge about species and serogroups causing human leptospirosis, especially in countries with low rates of culture sample collection or Leptospira isolation.

Phosphorus Removal from Aerobic Granular Sludge: Proliferation of Polyphosphate-Accumulating Organisms (PAOs) under Different Feeding Strategies

Aerobic granular sludge (AGS) is known for high phosphorus removal from wastewaters, and phosphorus can be recovered from high phosphorus-containing waste sludge granules. This study aimed at determining the feeding strategy that provides the best performance in terms of the proliferation of polyphosphate-accumulating organisms (PAOs) and phosphorus removal. Using three AGS bioreactors, this study compared phosphorus removal and the proliferation dynamics of PAOs under three different feeding strategies: anaerobic slow feeding (R1), pulse feeding + anaerobic mixing (R2), and pulse feeding (R3). Results indicate that R1 and R2 achieved significantly higher phosphorus removal (97.6 ± 3% for R1 and 98.3 ± 1% for R2) than R3 (55 ± 11%). The anaerobic slow feeding procedure (R1) achieved the highest specific phosphorus release rate (SPRR) and specific phosphorus uptake rate (SPUR) as compared to the other two feeding conditions. 16S ribosomal ribonucleic acid (rRNA) gene sequencing assay of the microbial community for the three feeding strategies indicated that although the feeding strategy impacted reactor performance, it did not significantly alter the microbial community. The bacteria community composition maintained a similar degree of diversity. Proteobacteria, Bacteroidetes, and Verrucomicrobia were the dominant bacterial phyla in the system. Dominant PAOs were from the class Betaproteobacteria and the genera Paracoccus and Thauera. Glycogen-accumulating organisms were significantly inhibited while other less-known bacteria such as Wandonia and Hyphomonas were observed in all three reactors.

Bacterial community assembly and beta-lactamase (bla) genes regulation in a full-scale chloraminated drinking water supply system

Beta-lactamases (bla) are the largest class of antibiotic resistance genes (ARGs) and can survive drinking water treatment processes. However, the key influencing factors of bla genes and the control measures in drinking water supply systems (DWSSs) remain unclear. Quantitative PCR, metagenomic, and 16S rRNA gene sequencing assays were performed to investigate the bla genes in the DWSS. The main carriers of bla genes are genera Caenimonas and Sphingomonas, which were the dominant bacterial genera in the DWSS. The abundance of bla genes was associated with bacterial community assembly events. When the heterotrophic plate count (HPC) in water was below 500 CFU/mL, stochastic assembly was the major contributor to bacterial community assembly and the bacterial community was less affected by the environment. When the HPC in water was above 500 CFU/mL, deterministic assembly played an important role and decreasing the organic matter improved the efficiency of bla genes control. Organic matter can affect bla genes by shifting the diversity of the microbial community, and less organic matter appeared to be beneficial to reducing the bacterial niche width in the DWSS. Nanofiltration (NF) can decrease the effluent organic matter in waterworks and slow the dissemination of bacteria carrying bla genes in the DWSS.

Improved ginseng production under continuous cropping through soil health reinforcement and rhizosphere microbial manipulation with biochar: a field study of Panax ginseng from Northeast China.

ABSTRACTThe production of ginseng, an important Chinese medicine crop, has been increasingly challenged by soil degradation and pathogenic disease under continuous cropping in Northeast China. In a field experiment, an Alfisol garden continuously cropped with Chinese ginseng (Panax ginseng C. A. Meyer) was treated with soil amendment at 20 t ha−1 with maize (MB) and wood (WB) biochar, respectively, compared to conventional manure compost (MC). Two years after the amendment, the rooted topsoil and ginseng plants were sampled. The changes in soil fertility and health, particularly in the soil microbial community and root disease incidence, and in ginseng growth and quality were portrayed using soil physico-chemical assays, biochemical assays of extracellular enzyme activities and gene sequencing assays as well as ginsenoside assays. Topsoil fertility was improved by 23% and 39%, ginseng root biomass increased by 25% and 27%, and root quality improved by 6% and 18% with WB and MB, respectively, compared to MC. In the ginseng rhizosphere, fungal abundance increased by 96% and 384%, with a significant and insignificant increase in bacterial abundance, respectively, under WB and MB. Specifically, the abundance of Fusarium spp. was significantly reduced by 19–35%, while that of Burkholderia spp. increased by folds under biochar amendments over MC. Relevantly, there was a significant decrease in the abundance proportion of pathotrophic fungi but a great increase in that of arbuscular mycorrhizal fungi, along with an enhanced microbial community network complexity, especially fungal community complexity, under biochar amendments. Thus, biochar, particularly from maize residue, could promote ginseng quality production while enhancing soil health and ecological services, including carbon sequestration, in continuously cropped fields.

The first reported outbreak of an undetermined species of human infection with spotted fever group Rickettsia in Lu'an, China

ObjectiveOn May 2, 2017, an outbreak of unexplained fever with rashes was reported in Lu'an, China. In this study, we aimed to identify the possible pathogens, epidemiological characteristics, and risk factors of this outbreak. MethodsWe conducted descriptive field epidemiological studies. Blood samples were tested using an indirect immunofluorescence assay for Rickettsia rickettsii antibody, and nested polymerase chain reaction and gene sequencing assays were performed. ResultsWe recruited 39 cases who had symptomatic onset from April 20 to June 8, 2017. Among these, 9 were suspected cases, 18 were probable cases, and 12 were confirmed cases. No one died. The main clinical manifestations were fever (100%), rash (100%), fatigue (97.3%), myalgia (83.8%), and anorexia (83.8%). None of the patients died. Thirty-seven patients who were treated with antibiotics during hospitalization showed significant improvement. The cases were distributed across 14 townships in 2 counties. The median age was 59 (43.0–81.0) years, of which 93.3% had a history of tea picking (28/30), and 77.3% (17/22) had a history of tick bites. The mean incubation period was 5.0 days (2.0–13.0 days). Serum IgG titers were higher in convalescent patients than in the general population (p = 0.016). Phylogenetic analysis revealed that the ompA sequences of Rickettsia sp. Lu'an-2018 had an 86.8%–99.0% sequence identity with the 23 strains of Rickettsia found worldwide. ConclusionsThis was the first reported outbreak of an undetermined species of a human infection with the spotted fever group of Rickettsia in China, which might be caused by ticks biting local residents when picking tea.

DNA metabarcoding-based diet survey for the Eurasian otter (Lutra lutra): Development of a Eurasian otter-specific blocking oligonucleotide for 12S rRNA gene sequencing for vertebrates.

The Eurasian otter (Lutra lutra) is an endangered species for which diet analyses are needed as part of its conservation efforts. Eurasian otters feed on vertebrates, such as fishes, and invertebrates, such as crustaceans, but their detailed taxonomies are not fully understood in part due to limited resolving power of traditional morphological identification methods. Here, we used high-throughput sequencing (HTS)-based DNA metabarcoding approaches to analyze diet profiles of Eurasian otters inhabiting a marshy estuary area in Korea. We investigated their diet profiles based on spraint sampling followed by DNA metabarcoding analyses targeting 12S rRNA gene region for vertebrates, 16S rRNA gene region for invertebrates, and cytochrome c oxidase 1 (COI) gene region for fishes. For the vertebrate analysis, a blocking oligonucleotide (OBS1) was designed to suppress amplification of DNA fragments derived from the otters. The 12S rRNA gene sequencing assay detected species belonging to fishes (95%) and amphibians (3.3%). Fishes detected by 12S rRNA gene sequencing included crucian carp (Carassius auratus), mullets (Mugil spp.), bluegill (Lepomis macrochirus), and northern snakehead (Channa argus), which were also detected by COI gene sequencing. Among invertebrates, mud flat crabs (Helicana spp.) and shrimps (Palaemon spp.) were abundant. The designed blocking oligonucleotide OBS1 effectively inhibited amplification of the otter's DNA, with only up to 0.21% of vertebrate sequence reads assigned to the otter. This study demonstrated that HTS-based DNA metabarcoding methods were useful to provide in-depth information regarding diet profiles of the otters at our sampling site. By using HTS-based DNA metabarcoding approaches, future research will explore detailed taxonomies of their diets across locations and seasons.

English

Hepatitis C virus is a serious hepatic disease that could be developed into hepatocellular carcinoma. Previously, some probiotic strains showed a natural therapeutic activity against the fatty liver disorder. Therefore, it could make sense to evaluate the antiviral and antibacterial responses to probiotics as Lactobacillus acidophilus and Bifidobacteria spp. in patients with chronic hepatitis C virus. Twenty (20) patients with chronic hepatitis C (both gender in age 47± 5 years) were treated with capsule that contains probiotics (L. acidophilus and Bifidobacterium spp.). They administered one capsule per day for a month before HCV treatment; blood and urine samples were collected before and after the given treatment and they were processed for a quantitative estimation of HCV by PCR, identification of bacteria by VITEK2 system and 16S r RNA gene sequencing assay. Moreover, the estimation of antibacterial activity of probiotics by antibiotic sensitivity test, counts of leukocytes, CD3+ T cells and CD56+ natural killer cells by flowcytometry, DGAT1 by ELISA. Administration of probitics enhanced the treatment response rate to HCV treatments pegylated IFN-α and ribavirin by 25% and showed an antibacterial activity against five species of the most common infectious bacteria in chronic HCV patients which were identified by this study. CD3+ cells counts and CD56+ natural killer cells were increased. L. acidophilus and Bifidobacterium spp. can act as a supportive supplement with antiviral and antibacterial activities; we recommend Probiotics side by side with HCV treatments.   Key words: Hepatitis C virus, Lactobacillus acidophilus and Bifidobacteria spp., CD3+, CD56+, pegylated IFN-α and ribavirin.

Doing more with less: fluorescence in situ hybridization and gene sequencing assays can be reliably performed on archival stained tumor tissue sections.

Little is known about molecular testing on tumor tissue retrieved from stained sections, for which there may be a clinical need. We retrospectively analyzed 112 sections from 56 tumor patients using either fluorescence in situ hybridization (FISH) with different probes (19 sections from 17 patients) or Sanger or targeted next generation sequencing for detection of BRAF, EGFR, KRAS, C-KIT, and TP53 mutations (93 sections from 39 patients). Tumor tissue sections had been stained by hematoxylin and eosin (H&E) (42 sections) or by immunohistochemistry for cytoplasmic or nuclear/nuclear-cytoplasmic markers (70 sections) with a peroxidase (P-IHC, with 3,3'-diaminobenzidine as chromogen) or alkaline phosphatase label (AP-IHC, with Warp Red™ as chromogen). For FISH analysis, the concordance rate between the original diagnosis and that obtained on H&E- or P-IHC-stained tissue sections (AP-IHC was not on record for this set of patients) was 95% (18 out of 19 tumor sections). Only one tumor sample, diffusely positive for MLH1, did not yield any nuclear hybridization signal. For sequencing analysis, the concordance rate was 100% on negative P-IHC and positive AP-IHC-stained sections, regardless of the subcellular localization of the reaction product. Mutations were detected in only 52% of cases expressing nuclear/nuclear-cytoplasmic markers, regardless of the sequencing technology used (p = 0.0002). In conclusion, stained sections may be a valuable resource for FISH or sequencing analysis, but on cases expressing nuclear markers sequencing results need to be interpreted cautiously.

Simplified Large-Scale Sanger Genome Sequencing for Influenza A/H3N2 Virus

BackgroundThe advent of next-generation sequencing technologies and the resultant lower costs of sequencing have enabled production of massive amounts of data, including the generation of full genome sequences of pathogens. However, the small genome size of the influenza virus arguably justifies the use of the more conventional Sanger sequencing technology which is still currently more readily available in most diagnostic laboratories.ResultsWe present a simplified Sanger-based genome sequencing method for sequencing the influenza A/H3N2 virus in a large-scale format. The entire genome sequencing was completed with 19 reverse transcription-polymerase chain reactions (RT-PCRs) and 39 sequencing reactions. This method was tested on 15 native clinical samples and 15 culture isolates, respectively, collected between 2009 and 2011. The 15 native clinical samples registered quantification cycle values ranging from 21.0 to 30.56, which were equivalent to 2.4×103–1.4×106 viral copies/µL of RNA extract. All the PCR-amplified products were sequenced directly without PCR product purification. Notably, high quality sequencing data up to 700 bp were generated for all the samples tested. The completed sequence covered 408,810 nucleotides in total, with 13,627 nucleotides per genome, attaining 100% coding completeness. Of all the bases produced, an average of 89.49% were Phred quality value 40 (QV40) bases (representing an accuracy of circa one miscall for every 10,000 bases) or higher, and an average of 93.46% were QV30 bases (one miscall every 1000 bases) or higher.ConclusionsThis sequencing protocol has been shown to be cost-effective and less labor-intensive in obtaining full influenza genomes. The constant high quality of sequences generated imparts confidence in extending the application of this non-purified amplicon sequencing approach to other gene sequencing assays, with appropriate use of suitably designed primers.

Development of a novel DNA sequencing method not only for hepatitis B virus genotyping but also for drug resistant mutation detection

BackgroundIn HBV-infected patients, different genotypes of the hepatitis B virus influence liver disease progression and response to antiviral therapy. Moreover, long-term antiviral therapy will eventually select for drug-resistant mutants. Detection of mutations associated to antiviral therapy and HBV genotyping are essential for monitoring treatment of chronic hepatitis B patients.ResultsIn this study, a simple method of partial-S gene sequencing using a common PCR amplification was established for genotyping clinical HBV isolates sensitively, which could detect the drug-resistant mutations successfully at the same time.ConclusionsThe partial S gene sequencing assay developed in this study has potential for application in HBV genotyping and drug resistant mutation detection. It is simpler and more convenient than traditional S gene sequencing, but has nearly the same sensitivity and specificity when compared to S gene sequencing.

A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis

The focus of the current research was to develop real-time PCR assays with improved sensitivity and the capacity to simultaneously speciate the 3 most common mycoplasma mastitis agents: Mycoplasma bovis, Mycoplasma californicum, and Mycoplasma bovigenitalium. Real-time PCR was chosen because it provides rapid results. Partial 16S rRNA gene sequencing was used as the gold standard for evaluating candidate real-time PCR assays. To ascertain the real-time PCR assay specificity, reference strains of Mycoplasma species, Acholeplasma axanthum, and common gram-positive and gram-negative mastitis pathogens were tested. No cross-reactions were observed. Mycoplasma spp. isolated from bovine milk samples (n=228) and other organ sites (n=40) were tested by the real-time PCR assays and the partial 16S rRNA gene sequencing assay. Overall accuracy of this novel real-time PCR was 98.51%; 4 of 228 isolates identified as M. bovis by the partial 16S rRNA gene sequencing assay were identified as both M. bovis and M. californicum by real-time PCR. Subsequent amplicon sequencing suggested the presence of both M. bovis and M. californicum in these 4 samples. Using a cycle threshold of 37, the detection limits for real-time PCR were 10 copies of DNA template for both M. bovis and M. bovigenitalium, and 1 copy for M. californicum. This real-time PCR assay is a diagnostic technique that may be used as a screening tool or as a confirmation test for mycoplasma mastitis.

The importance of β globin deletion analysis in the evaluation of patients with β thalassemia

Beta globin deletion/duplication analysis may serve as a useful adjunct to sequence analysis. Our purpose was to develop a robust assay for beta globin deletion/duplication analysis and determine its role in evaluating patients with beta thalassemia. A single tube semi-quantitative fluorescent PCR assay capable of detecting deletions and duplications in the beta globin cluster and the associated locus control region (LCR) was developed and validated. Six hundred seventy one de-identified samples submitted for beta globin sequence analysis were tested for deletions and duplications of the beta globin cluster. Twenty-two deletions were detected (3%, 22/671). Seventeen of the 22 (82%) deletion samples were negative for mutations in the whole gene sequencing assay. For 5 of the samples, homozygous point mutations were inferred by beta globin sequencing. Among the deletions detected, 11 (50%) involved only the beta globin gene (5 covering the entire gene, 2 spanning the 5' end of the gene and 4 encompassing the 3' end of the gene). Ten samples (45%) were heterozygous delta-beta deletions spanning both the delta globin and beta globin genes. One patient with a single deletion had Hb Lepore. Beta globin deletion/duplication analysis is necessary to correctly identify the genotype in some patients being evaluated for beta thalassemia.

Comprehensive and EfficientHBBMutation Analysis for Detection of β-Hemoglobinopathies in a Pan-Ethnic Population

Current methods that assay hemoglobin beta-globin chain variants can have limited clinical sensitivity when applied techniques identify only a predefined panel of mutations. Even sequence-based assays may be limited depending on which gene regions are investigated. We sought to develop a clinically practical yet inclusive molecular assay to identify beta-globin mutations in multicultural populations. We highlight the beta-globin mutation detection assay (beta-GMDA), an extensive gene sequencing assay. The polymerase chain reaction (PCR) primers are located to encompass virtually all hemoglobin beta locus (HBB) mutations. In addition, this assay is able to detect, by gap PCR, a common large deletion (Delta619 base pair), which would be missed by sequencing alone. We describe our 5-year experience with the beta-GMDA and indicate its capability for detecting homozygous, heterozygous, and compound heterozygous sequence changes, including previously unknown HBB variants. The beta-GMDA offers superior sensitivity and ease of use with comprehensive detection of HBB mutations that result in beta-globin chain variants.

Improving Accuracy of Tay Sachs Carrier Screening of the Non-Jewish Population: Analysis of 34 Carriers and Six Late-Onset Patients With HEXA Enzyme and DNA Sequence Analysis

The purpose of this study was to determine whether combining different testing modalities namely beta-hexosaminidase A (HEXA) enzyme analysis, HEXA DNA common mutation assay, and HEXA gene sequencing could improve the sensitivity for carrier detection in non-Ashkenazi (AJ) individuals. We performed a HEXA gene sequencing assay, a HEXA DNA common mutation assay, and a HEXA enzyme assay on 34 self-reported Tay-Sachs disease (TSD) carriers, six late-onset patients with TSD, and one pseudodeficiency allele carrier. Sensitivity of TSD carrier detection was 91% for gene sequencing compared with 91% for the enzyme assay and 52% for the DNA mutation assay. Gene sequencing combined with enzyme testing had the highest sensitivity (100%) for carrier detection. Gene sequencing detected four novel mutations, three of which are predicted to be disease causing [118.delT, 965A-->T (D322V), and 775A-->G (T259A)]. Gene sequencing is useful in identifying rare mutations in patients with TSD and their families, in evaluating spouses of known carriers for TSD who have indeterminate enzyme analysis and negative for common mutation analysis, and in resolving ambiguous enzyme testing results.

Expression of Periostin and Identification of Periostin-Isoforms in Colorectal Carcinoma

Periostin, a gene that is initially associated with bone and tooth development, is linked to tumourigenesis of colorectal carcinoma (CRC). Increasing evidence via functional study revealed this protein as an oncogenic promoter or, in certain circumstances, as a tumour suppressor. Therefore, in order to verify the role of periostin in CRC, we performed gene expression study using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis in local and commercially available colorectal cancer samples. Expression of periostin mRNA was detected at a significantly higher level in the tumour specimens (P < 0.05), suggesting this gene as a putative oncogene. Further studies using PCR and gene sequencing assays revealed the presence of ten periostin isoforms that harbour deletion at specific C-terminus region, possibly caused by alternative splicing events. Three isoforms were identical to published data of periostin (GenBank), while the remaining variants that have not been reported may provide new information on the genetics of colon cancer.

Detection of mutations in the hepatitis B virus polymerase gene.

Antiviral treatment of chronic hepatitis B infection aims to reduce viral replication and/or to affect the immune response to the virus and virus-infected cells. The development of reverse transcriptase inhibitors such as lamivudine, which has been shown to be a safe and potent inhibitor of hepatitis B virus (HBV) replication (1)(2), has facilitated major advances in the antiviral treatment of chronic hepatitis B. Today, lamivudine is a first-line therapy for prophylaxis of HBV recurrence in decompensated cirrhotic patients and liver transplant recipients. A major problem with lamivudine treatment is the emergence of drug resistance, which increases with extended duration of therapy (3). Resistant variants have been localized in the reverse transcriptase ( rt ) region of the HBV polymerase gene. Lamivudine-resistant amino acids have been described at positions rt180 (rtL180M) and rt204 (rtM204V/I/S) (4)(5). Methods for the identification of mutations in the HBV polymerase gene include conventional DNA sequencing, restriction fragment length polymorphism analysis, and reverse hybridization (6)(7). In the past, conventional direct DNA sequencing, which is the gold standard method, was the most labor-intensive and time-consuming method (6). Recently, however, a standardized and largely automated HBV polymerase gene-sequencing assay, the TrugeneTM HBV Genotyping Kit, version 1.0 (Bayer/Visible Genetics, Toronto, Ontario), became commercially available. This assay may be suitable for routine diagnostic laboratory work and clinical trial applications. In this preliminary study, we evaluated the performance of the new HBV genotyping assay. Patients undergoing lamivudine treatment were retrospectively investigated for emergence of specific mutations. Serum samples from five HBV DNA-positive (serum load >1000 HBV DNA copies/mL) patients undergoing lamivudine therapy for more than 6 months were analyzed retrospectively. Blood had been collected in 9.0-mL tubes (VacuetteTM; Greiner Bio-one GmbH), and after centrifugation, sera had been aliquoted and stored at −70 °C. Alanine aminotransferase …