scholarly journals Comparative Genomics of Mycoplasma synoviae and New Targets for Molecular Diagnostics

2021 ◽  
Vol 8 ◽  
Author(s):  
Bin Xu ◽  
Xi Chen ◽  
Fengying Lu ◽  
Yu Sun ◽  
Huawei Sun ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Diagnostic ◽  
Economic Losses ◽  
Clinical Samples ◽  
Comparative Genomic ◽  
Evolutionary Analysis ◽  
Genome Sequences ◽  
Mycoplasma Synoviae

Mycoplasma synoviae is an important pathogen of poultry, causing significant economic losses in this industry. Analysis of the unique genes and shared genes among different M. synoviae strains and among related species is helpful for studying the molecular pathogenesis of M. synoviae and provides valuable molecular diagnostic targets to facilitate the identification of M. synoviae species. We selected a total of 46 strains, including six M. synoviae strains, from 25 major animal (including avian) Mycoplasma species/subspecies that had complete genome sequences and annotation information published in GenBank, and used them for comparative genomic analysis. After analysis, 16 common genes were found in the 46 strains. Thirteen single-copy core genes and the 16s rRNA genes were used for genetic evolutionary analysis. M. synoviae was found to have a distant evolutionary relationship not only with other arthritis-causing mycoplasmas, but also with another major avian pathogen, Mycoplasma gallisepticum, that shares the major virulence factor vlhA with M. synoviae. Subsequently, six unique coding genes were identified as shared among these M. synoviae strains that are absent in other species with published genome sequences. Two of the genes were found to be located in the genetically stable regions of the genomes of M. synoviae and were determined to be present in all M. synoviae isolated strains (n = 20) and M. synoviae-positive clinical samples (n = 48) preserved in our laboratory. These two genes were used as molecular diagnostic targets for which SYBR green quantitative PCR detection methods were designed. The two quantitative PCR methods exhibited good reproducibility and high specificity when tested on positive plasmid controls and genomic DNA extracted from different M. synoviae strains, other major avian pathogenic bacteria/mycoplasmas, and low pathogenic Mycoplasma species. The detection limit for the two genes was 10 copies or less per reaction. The clinical sensitivity and specificity of the quantitative PCR methods were both 100% based on testing chicken hock joint samples with positive or negative M. synoviae infection. This research provides a foundation for the study of species-specific differences and molecular diagnosis of M. synoviae.

scholarly journals Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

2002 ◽  
Vol 68 (10) ◽  
pp. 5064-5081 ◽  
Author(s):  
Alexander Loy ◽  
Angelika Lehner ◽  
Natuschka Lee ◽  
Justyna Adamczyk ◽  
Harald Meier ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Amplification ◽  
Oligonucleotide Microarray ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Sulfate Reducing ◽  
Specific Pcr

ABSTRACT For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

scholarly journals Effect of gamma irradiation on viability and DNA of Staphylococcus epidermidis and Escherichia coli

2006 ◽  
Vol 55 (9) ◽  
pp. 1271-1275 ◽  
Author(s):  
Andrej Trampuz ◽  
Kerryl E. Piper ◽  
James M. Steckelberg ◽  
Robin Patel
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Gamma Irradiation ◽  
Staphylococcus Epidermidis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Techniques ◽  
Bacterial Cells ◽  
E Coli

Gamma irradiation is widely used for sterilization; however, its effect on elimination of amplifiable DNA, an issue of relevance to molecular diagnostic approaches, has not been well studied. The effect of gamma irradiation on the viability of Staphylococcus epidermidis and Escherichia coli (using quantitative cultures) and on their DNA (using quantitative 16S rRNA gene PCR) was evaluated. Viability was abrogated at 2.8 and 3.6 kGy for S. epidermidis and E. coli, respectively. The radiation dose required to reduce viable bacteria by one log10 (D 10 value) was 0.31 and 0.35 kGy for S. epidermidis and E. coli, respectively. D 10 values for amplifiable DNA extracted from bacteria were 2.58 and 3.09 kGy for S. epidermidis and E. coli, respectively, whereas D 10 values for amplifiable DNA were significantly higher for DNA extracted from irradiated viable bacterial cells (22.9 and 52.6 kGy for S. epidermidis and E. coli, respectively; P<0.001). This study showed that gamma irradiation of DNA in viable bacterial cells has little effect on amplifiable DNA, was not able to eliminate amplifiable 16S rRNA genes at a dose of up to 12 kGy and cannot therefore be used for elimination of DNA contamination of PCR reaction components or laboratory equipment when this DNA is present in microbial cells. This finding has practical implications for those using molecular diagnostic techniques in microbiology.

scholarly journals Quantitative and Qualitative Characterization of Plant Endo-bacteriome by Plant Dna-free Sequencing Method

2020 ◽  
Author(s):  
Lili Zhang ◽  
Liying Chen ◽  
Mengting Zhang ◽  
Da Liu ◽  
Hongbo Sun ◽  
...  
Keyword(s):  
16S Rrna ◽  
Quantitative Pcr ◽  
Endophytic Bacteria ◽  
Plant Material ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Variable Regions ◽  
Plant Dna ◽  
Primer Sets

Abstract Background: Plant endophytic bacteria colonize plants’ internal tissues and interact with plants more closely than do epiphytic or environmental bacteria. However, the community structure of such endophytic bacteria could not be efficiently deciphered, and the microbiota abundance could not be quantified through absolute quantification. Application of 16S rRNA gene sequencing to characterize the plant endophytic community is greatly hindered by the high sequence identities among bacterial 16S rRNA, plant mitochondrial 18S rRNA and chloroplast 16S rRNA genes. This makes it difficult to identify bacterial sequences among total DNAs extracted from plant material.Results: We designed PCR primer sets that are able to specifically amplify bacterial DNAs, even when there are very few bacteria colonizing the plant material. We computationally and experimentally evaluated the specificity, coverage, and accuracy of the newly designed primer sets (322F/796R and 799F/1107R) and two widely used primer sets (338F/806R and 799F/1193R). When applied to a same planting-soil community through next generation sequencing (NGS), the four different primer sets revealed similar high-abundant taxa composition with variation in taxa abundance. Primer sets amplifying the same 16S variable regions generated more comparable sequencing results. When applied to a rice endo-bacteriome, both 799F/1107R and modified 322F-Dr/796Rs (Primer pair 322F/796R with a penultimate-base substitution in 322F) produced plant DNA-free bacterial amplicon libraries. Primer 322F-A/796R was then used through NGS to decipher the rice endo-bacteriomes. The rice root and leaf endo-bacteriomes shared 66.36% OTU identity but enriched different bacterial species. Within the same host genotype and soil type, the root endo-bacteriome was more stable than the leaf endo-bacteriome across individual plants. 322F-A/796R was used through absolute quantitative PCR to quantitate the population size of leaf or root endophytes, which revealed 106–107 and 109–1010 bacteria per gram fresh weight, respectively.Conclusions: This is the first study to develop plant DNA-free bacterial 16S amplification methods. The newly designed primer sets combined with NGS deciphered the rice endo-bacteriome structure, and absolute quantitative PCR quantitated the size of the endobacterial population. The protocols developed here are suitable for various plants, will significantly advance studies on plant endo-bacteriomes.

scholarly journals An Omics Approach to Diagnosing or Investigating Fungal Keratitis

2019 ◽  
Vol 20 (15) ◽  
pp. 3631 ◽  
Author(s):  
Ming-Tse Kuo ◽  
Jiunn-Liang Chen ◽  
Shiuh-Liang Hsu ◽  
Alexander Chen ◽  
Huey-Ling You
Keyword(s):  
Dynamic Change ◽  
Diagnostic Value ◽  
Fungal Keratitis ◽  
Internal Transcribed Spacers ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Clinical Samples ◽  
Clinical Practices ◽  
Proteomic Approach ◽  
Clinical Diagnoses

Fungal keratitis (FK) is one of the most severe corneal infectious diseases. FK often leads to poor visual prognosis and thus requires accurate diagnosis. Conventional approaches, including clinical diagnoses, smears, and cultures, often fail to provide reliable diagnostic value. Omics approaches, such as those using genomic, metagenomic, and tear proteomic data sources, provide promising features for improving the diagnosis and monitoring the progression of FK. Genomic approaches are based mainly on detecting amplicons of ribosomal RNA genes, and internal transcribed spacers are gradually gaining popularity in clinical practices. A metagenomic approach based on 16S rRNA genes may help monitor the dynamic change of conjunctival microbiota associated with an FK event, whereas that based on shot-gun and 18S rRNA target enrichment sequencing could have the potential to diagnose FK using clinical samples. A tear proteomic approach may provide comprehensive information about ocular surface defense and injury during FK. Representative up- and down-regulated proteins during FK could also be used as biomarkers to determine the clinical course and develop a treatment strategy in different stages of FK. Consequently, a personalized tear proteomic approach will soon play a key role in FK management.

scholarly journals Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples

2012 ◽  
Vol 78 (12) ◽  
pp. 4481-4489 ◽  
Author(s):  
Robert Brankatschk ◽  
Natacha Bodenhausen ◽  
Josef Zeyer ◽  
Helmut Bürgmann
Keyword(s):  
Microbial Community ◽  
Quantitative Pcr ◽  
Environmental Samples ◽  
Absolute Quantification ◽  
Geobacter Sulfurreducens ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gene Copy ◽  
Nostoc Commune ◽  
Content Type

ABSTRACTReal-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting thenifHand 16S rRNA genes. TheEvalues of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in theirEvalues, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences inEand was derived from the ΔΔCTmethod with correction forE, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures fromGeobacter sulfurreducens(DSM 12127) andNostoc commune(Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in theirEvalues. While the SC method deviated from the expectednifHgene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences inEbetween the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.

scholarly journals Life Cycle and Genetic Identification of Argas persicus Infesting Domestic Fowl in Khyber Pakhtunkhwa, Pakistan

2021 ◽  
Vol 8 ◽  
Author(s):  
Hafsa Zahid ◽  
Sebastián Muñoz-Leal ◽  
Muhammad Qayash Khan ◽  
Abdulaziz S. Alouffi ◽  
Marcelo B. Labruna ◽  
...  
Keyword(s):  
Life Cycle ◽  
Domestic Fowl ◽  
The United States ◽  
Genetic Identification ◽  
16S Rrna Genes ◽  
Health Concern ◽  
Rrna Genes ◽  
Economic Losses ◽  
Local Alignment ◽  
Argas Persicus

Ticks transmit numerous pathogens to animals including humans; therefore, they are parasites of health concern. Soft ticks infesting domestic fowl in Pakistan are carriers of viruses and bacteria and cause unestimated economic losses in the poultry sector. The current study was intended to identify soft ticks infesting domestic fowl and understand their spatiotemporal distribution along 1 year. A sum of 7,219 soft ticks were collected from 608 domestic fowl in 58 infested shelters; 938 (12.9%) ticks were found on the host and 6,281 (87%) in the shelters. The collected ticks comprised 3,503 (48.52%) adults including 1,547 (21.42%) males and 1,956 (27.09%) females, 3,238 (44.85%) nymphs, and 478 (6.62%) larvae. The most prevalent life stages were adults, followed by nymphs and larvae. Overall tick prevalence considering all visited shelters was 38.66% (58/150). The highest tick prevalence was found in district Lakki Marwat (50.03%) followed by Peshawar (31.08%) and Chitral (18.88%) districts. All ticks were morpho-taxonomically identified as Argas persicus. To determine their life cycle, adult A. persicus were reared in the laboratory infesting domestic fowl (Gallus gallus domesticus). The life cycle was completed in 113–132 days (egg to egg) with a mean temperature of 33 ± 3°C and relative humidity of 65 ± 5%. Individual ticks were used for DNA extraction and subjected to polymerase chain reaction (PCR) using specific primers for the amplification of a partial fragment of mitochondrial cytochrome oxidase subunit I (cox1) and 16S ribosomal RNA (16S rRNA) genes. Obtained amplicons were compared using basic local alignment search tool (BLAST) to scan for homologous sequences. Phylogenetic trees showed A. persicus from Pakistan clustering with conspecific sequences reported from Australia, Chile, China, Kenya, and the United States. This is the first study aiming to reproduce the life cycle of A. persicus and genetically identify this tick in the region. Further studies are encouraged to investigate the pathogens associated with this soft tick species in Pakistan.

Avian Mycoplasma gallisepticum and Mycoplasma synoviae: Advances in diagnosis and control

2021 ◽  
Vol 1 (2) ◽  
pp. 46-55
Author(s):  
Abdelrahman A. Abdelrahman ◽  
Salama A. S. Shany ◽  
Mansy A. A. Dardeer ◽  
Kareem E. Hassan ◽  
Ahmed Ali ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Mycoplasma Gallisepticum ◽  
Current Control ◽  
Molecular Diagnostic ◽  
Economic Losses ◽  
Mycoplasma Infection ◽  
Chain Reaction ◽  
Mycoplasma Synoviae ◽  
Polymerase Chain ◽  
And Control

Both of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) infections are the most common Mycoplasma infection in domestic poultry. The disease is associated with economic losses in poultry. MG and MS are commonly spread within chickens (Gallus gallus domesticus) and turkeys (Meleagris gallopavo domesticus) flocks; however, they are frequently isolated from quails (Coturnix coturnix) and several avian species. Diagnosis of MG or MS infections is confirmed by isolating the organism in a cell-free medium or directly detecting its DNA in infected tissues or swab samples. Serological tests are also widely used for diagnosis. However, advances in molecular biology represented a rapid and sensitive alternative to the traditional culture methods requiring specialized techniques and sophisticated reagents. Several Mycoplasma molecular diagnostic tests are implemented: including polymerase chain reaction (PCR), Random Amplified Polymorphic DNA (RAPD), arbitrary primed polymerase chain reactions (AP‐PCR), and Multiplex real-time polymerase chain reaction (Multiplex MGMS). Current control practices against Mycoplasma infection include intense biosecurity, biosurveillance, medication, and vaccination. However, the egg-borne nature of avian Mycoplasma infection complicates controlling the infection. This review focuses on the advances in diagnosis and control of avian Mycoplasma infection, especially MG and MS infections.

scholarly journals Correlation between Composition of the Bacterial Community and Concentration of Volatile Fatty Acids in the Rumen during the Transition Period and Ketosis in Dairy Cows

2012 ◽  
Vol 78 (7) ◽  
pp. 2386-2392 ◽  
Author(s):  
Xiaoxu Wang ◽  
Xiaobing Li ◽  
Chenxu Zhao ◽  
Pan Hu ◽  
Hui Chen ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Quantitative Pcr ◽  
Volatile Fatty Acids ◽  
Transition Period ◽  
Negative Energy ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Selenomonas Ruminantium ◽  
Megasphaera Elsdenii

ABSTRACTThe transition period is a severe challenge to dairy cows. Glucose supply cannot meet demand and body fat is mobilized, potentially leading to negative energy balance (NEB), ketosis, or fatty liver. Propionate produces glucose by gluconeogenesis, which depends heavily on the number and species of microbes. In the present study, we analyzed the rumen microbiome composition of cows in the transition period, cows with ketosis, and nonperinatal cows by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes and quantitative PCR. TRFLP analysis indicated that the quantity ofVeillonellaceaeorganisms was reduced and that ofStreptococcaceaeorganisms was increased in rumen samples from the transition period and ketosis groups, with the number ofLactobacillaceaeorganisms increased after calving. Quantitative PCR data suggested that the numbers of the main propionate-producing microbes,Megasphaera elsdeniiandSelenomonas ruminantium, were decreased, while numbers of the main lactate-producing bacterium,Streptococcus bovis, were increased in the rumen of cows from the transition period and ketosis groups, with the number ofLactobacillussp. organisms increased after calving. Volatile fatty acid (VFA) and glucose concentrations were decreased, but the lactic acid concentration was increased, in rumen samples from the transition period and ketosis groups. Our results indicate that the VFA concentration is significantly related to the numbers ofSelenomonas ruminantiumandMegasphaera elsdeniiorganisms in the rumen.

scholarly journals A comprehensive global perspective on phylogenomics and evolutionary dynamics of Small ruminant morbillivirus

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Muhammad Zubair Shabbir ◽  
Aziz-ul Rahman ◽  
Muhammad Munir
Keyword(s):  
Complete Genome ◽  
Evolutionary Dynamics ◽  
Small Ruminant ◽  
Evolutionary Relationship ◽  
Global Perspective ◽  
Comparative Genomic ◽  
Evolutionary Analysis ◽  
Genome Sequences ◽  
Individual Genome ◽  
Whole Genomes

AbstractA string of complete genome sequences of Small ruminant morbillivirus (SRMV) have been reported from different parts of the globe including Asia, Africa and the Middle East. Despite individual genome sequence-based analysis, there is a paucity of comparative genomic and evolutionary analysis to provide overarching and comprehensive evolutionary insights. Therefore, we first enriched the existing database of complete genome sequences of SRMVs with Pakistan-originated strains and then explored overall nucleotide diversity, genomic and residue characteristics, and deduced an evolutionary relationship among strains representing a diverse geographical region worldwide. The average number of pairwise nucleotide differences among the whole genomes was found to be 788.690 with a diversity in nucleotide sequences (0.04889 ± S.D. 0.00468) and haplotype variance (0.00001). The RNA-dependent-RNA polymerase (L) gene revealed phylogenetic relationship among SRMVs in a pattern similar to those of complete genome and the nucleoprotein (N) gene. Therefore, we propose another useful molecular marker that may be employed for future epidemiological investigations. Based on evolutionary analysis, the mean evolution rate for the complete genome, N, P, M, F, H and L genes of SRMV was estimated to be 9.953 × 10–4, 1.1 × 10–3, 1.23 × 10–3, 2.56 × 10–3, 2.01 × 10–3, 1.47 × 10–3 and 9.75 × 10–4 substitutions per site per year, respectively. A recombinant event was observed in a Pakistan-originated strain (KY967608) revealing Indian strains as major (98.1%, KR140086) and minor parents (99.8%, KT860064). Taken together, outcomes of the study augment our knowledge and current understanding towards ongoing phylogenomic and evolutionary dynamics for better comprehensions of SRMVs and effective disease control interventions.

scholarly journals Prevalence of Arcobacter and Other Pathogenic Bacteria in River Water in Nepal

Water ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 1416 ◽  
Author(s):  
Rajani Ghaju Shrestha ◽  
Sarmila Tandukar ◽  
Dinesh Bhandari ◽  
Samendra P. Sherchan ◽  
Yasuhiro Tanaka ◽  
...  
Keyword(s):  
16S Rrna ◽  
River Water ◽  
Quantitative Pcr ◽  
Water Samples ◽  
Pathogenic Bacteria ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
One Year ◽  
Bagmati River ◽  
Pcr Targeting

This study aims to determine the diversity of pathogenic bacteria in the Bagmati River, Nepal, during a one-year period. A total of 18 river water samples were collected from three sites (n = 6 per site) along the river. Bacterial DNA, which were extracted from the water samples, were analyzed for bacterial 16S rRNA genes by next-generation sequencing for 13 of 18 samples, and by quantitative PCR targeting Arcobacter for all 18 samples. The 16S rRNA sequencing identified an average of 97,412 ± 35,909 sequences/sample, which were then categorized into 28 phyla, 61 classes, and 709 bacterial genera. Eighteen (16%) genera of 111 potential pathogenic bacteria were detected with abundance ratios of >1%; Arcobacter, Acinetobacter, and Prevotella were the dominant genera. The Arcobacter abundance ratios were 28.6% (n = 1), 31.3 ± 15.8% (n = 6), and 31.8 ± 17.2% (n = 6) at the upstream, midstream, and downstream sites, respectively. Arcobacter was detected in 14 (78%) of 18 samples tested, with concentrations ranging from 6.7 to 10.7 log10 copies/100 mL, based on quantitative PCR. Our results demonstrate the poor bacterial quality of the Bagmati River water, suggesting a need for implementing more measures to reduce fecal contamination in the river water.

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