scholarly journals Comparison of Leifsonia xyli subsp. xyli molecular detection in heat-treated sugarcane setts1

2019 ◽  
Vol 49 ◽  
Author(s):  
Vanessa Duarte Dias ◽  
Renato Carrer Filho ◽  
Marcos Gomes da Cunha
Keyword(s):  
Nested Pcr ◽  
Bacterial Population ◽  
Positive Control ◽  
Molecular Techniques ◽  
Conventional Pcr ◽  
Bacterial Dna ◽  
Heat Treated ◽  
Pcr Method ◽  
Management Method ◽  
Primary Management

ABSTRACT The thermotherapy of sugarcane setts is currently the primary management method for Leifsonia xyli subsp. xyli (Lxx), in Brazil. When they are immersed, the enzymes and proteins of the bacterial cell are denatured without harming the setts buds. Due to possible escapes from detection and consequent bacterium survival to thermotherapy, what may result in asymptomatic seedlings, this study aimed to detect the Leifsonia xyli subsp. xyli bacterium in sugarcane setts using molecular techniques and different time and temperature combinations, with or without the addition of antibiotics. The conventional PCR method detected the Lxx bacterial DNA only in the positive control, consisting of a highly susceptible plant with a high bacterial concentration. Using the nested-PCR, the Lxx DNA was detected in all the treatments used. Thus, none of the treatments adopted in the thermotherapy was able to eliminate the Lxx from the setts, and the use of kasugamicin also did not eliminate the bacterium, but reduced the bacterial population in the tested treatments. These results confirm that the nested-PCR is a useful tool to detect the presence of this phytobacterium in setts that will be used as seedlings.

Estimating Giardia cyst viability using RT-PCR

2002 ◽  
Vol 2 (3) ◽  
pp. 107-115 ◽  
Author(s):  
M. Maux ◽  
I. Bertrand ◽  
C. Gantzer ◽  
J. Schwartzbrod
Keyword(s):  
Nested Pcr ◽  
Target Genes ◽  
Molecular Techniques ◽  
Giardia Cyst ◽  
Health Issues ◽  
Structural Protein ◽  
Gene Encoding ◽  
Response To Stress ◽  
Pcr Method ◽  
Giardia Cysts

The aim of this work was to evaluate the use of molecular techniques for the detection of viable Giardia cysts in the environment to assess public health issues. Three target genes were selected: the heat shock protein gene, HSP70, which is expressed in response to stress; the giardin gene, which encodes a structural protein; and, alcohol dehydrogenase E (ADHE), a novel gene encoding an enzyme involved in the metabolism of energy. We tested the efficiency of five protocols for the extraction of either genomic DNA or total RNA from Giardia cysts: two of these protocols were previously cited in the literature and three consisted of commercial DNA extraction kits. The brands of enzyme were determined according to the primers chosen and the amplification conditions were optimised: 2.5 mM MgCl2, 0.5 mM primers and 60°C for annealing temperature. A semi-nested PCR method and an RT semi-nested PCR procedure were developed to detect mRNA from these three genes and to estimate the viability of Giardia cysts.

scholarly journals The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles

2020 ◽  
Vol 3 (2) ◽  
pp. 39 ◽  
Author(s):  
Anna Ojo-Okunola ◽  
Shantelle Claassen-Weitz ◽  
Kilaza S. Mwaikono ◽  
Sugnet Gardner-Lubbe ◽  
Heather J. Zar ◽  
...  
Keyword(s):  
Human Milk ◽  
Dna Extraction ◽  
Bacterial Population ◽  
Illumina Miseq ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Dna Recovery ◽  
Culture Independent ◽  
Zymo Research

Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp® DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of Enhydrobacter and Acinetobacter. DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa.

scholarly journals Application of in situ PCR for the Detection of Bovine Leukaemia Virus (BLV) Infection in Dendritic Cell cultures

2014 ◽  
Vol 58 (3) ◽  
pp. 347-352 ◽  
Author(s):  
Ewelina Iwan ◽  
Maria Szczotka ◽  
Jacek Kuźmak
Keyword(s):  
Cell Cultures ◽  
Nested Pcr ◽  
Leukemia Virus ◽  
Positive Control ◽  
Adherent Cell ◽  
Bovine Leukaemia Virus ◽  
Leukaemia Virus ◽  
Pcr Method

Abstract The aim of the study was to develop an in situ PCR (IS-PCR) method for detection of bovine leukemia virus (BLV) in cell cultures. Samples from five BLV positive and five BLV negative cows were collected and dendritic cells (DCs) from blood, bone marrow, spleen, and lymph node were cultured. Cultures prepared from healthy animals were infected with BLV. After two weeks, the cells were tested by nested PCR and IS-PCR for the presence of proviral DNA. As a positive control adherent cell line permanently infected with BLV was used. BLV was successfully detected by IS-PCR in DCs from naturally infected cattle and DCs infected in vitro. In control, non-infected DCs, the results of the reaction were negative. The results of provirus detection by IS-PCR were similar with these performed with nested PCR. Additionally, IS-PCR provides many advantages, like specific localisation of infection and smaller number of cells needed as template for PCR.

scholarly journals Improved Sensitivity in Detection of FMS-like Tyrosine Kinase Internal Tandem Duplication of a Method Using Next-Generation Sequencing Data

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2844-2844
Author(s):  
Junghoon Shin ◽  
Daeyoon Kim ◽  
Yoojin Hong ◽  
Youngil Koh ◽  
Hongseok Yun ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Nested Pcr ◽  
Tandem Duplication ◽  
Conflicts Of Interest ◽  
Sensitive Detection ◽  
Detection Methods ◽  
Next Generation Sequencing Data ◽  
Conventional Pcr ◽  
Internal Tandem Duplication ◽  
Pcr Method

Abstract We developed new ITD detection algorithm (ITDetect) based on whole exome sequencing (WES) data for FMS-like tyrosine kinase internal tandem duplication (FLT3-ITD). ITDetect is based on BWA and is specified for ITD detection including FLT3-ITD. We validated and compared result of ITDetect with other ITD detecting algorithms using nested polymerase chain reaction (PCR) method. Nested PCR uses two types of primer specified for FLT3-ITD detection. In 81 acute myeloid leukemia patients with WES data, FLT3-ITD was positive in 11 patients (13%) when called with ITDetect, all of whom were validated with nested PCR. Meanwhile FLT3-ITD was positive only in 7/81 patients by conventional PCR. The concordance rate of ITDetect and nested PCR was 95% (77/81). ITDetect showed better ITD detection performance when compared with previously reported ITD callers. In large AML cohort (n=213), patients who were positive for FLT3-ITD with nested-PCR but not with conventional PCR had shorter survival outcomes than patients who were negative for FLT3-ITD with nested PCR, suggesting clinical significance of sensitive FLT3-ITD detection. In conclusion, we developed more sensitive detection methods for FLT3-ITD based on WES data that is clinically meaningful. Utilization of more sensitive detection method than conventional PCR in clinic should be considered. Figure FLT3-ITD detection procedure. Figure. FLT3-ITD detection procedure. Figure Performance of various NGS ITD detectors. Figure. Performance of various NGS ITD detectors. Figure Survival comparison between patients positive for FLT3-ITD by nested PCR method but negative by conventional PCR method and those negative by both methods. Figure. Survival comparison between patients positive for FLT3-ITD by nested PCR method but negative by conventional PCR method and those negative by both methods. Disclosures No relevant conflicts of interest to declare.

scholarly journals Salmonellosis impacts the proportions of faecal microbial populations in domestic cats fed 1–3-d-old chicks

2014 ◽  
Vol 3 ◽  
Author(s):  
K. R. Kerr ◽  
S. E. Dowd ◽  
K. S. Swanson
Keyword(s):  
Clinical Symptoms ◽  
Clinical Status ◽  
Microbial Populations ◽  
Domestic Cats ◽  
Negative Data ◽  
Bacterial Dna ◽  
Faecal Samples ◽  
Pcr Method ◽  
Post Hoc ◽  
Pyrosequencing Method

AbstractThere has been a recent increase in the feeding of unconventional diets, including whole-prey diets, to domestic pet cats. Our objective was to characterise faecal microbial populations of domestic cats fed whole and ground (6·35 mm grind) raw 1–3-d-old chicks (Rodent Pro). Faecal samples were collected from neutered male domestic cats (mean age = 5·7 years) fed these diet items in a crossover design. Bacterial DNA was isolated from faecal samples and amplicons of the 16S rRNA V4–V6 region were generated and analysed by 454 pyrosequencing. Faecal microbial populations of cats fed whole v. ground chicks did not differ. During the study, three cats presented with symptoms of infection (anorexia or diarrhoea) and tested clinically positive for Salmonella using a standard PCR method. The remaining cats tested negative. Data were analysed post hoc to test for differences in microbial populations due to clinical status. The predominant genera were Clostridium (9–30 %), unidentified Lachnospiraceae (10–28 %), Blautia (4–19 %), Peptococcus (2–19 %) and Fusobacterium (2–14 %). Faeces of cats testing clinically positive for Salmonella had higher (P ≤ 0·05) proportions of the genera Coprococcus (5·6 v. 0·4 %) and Escherichia (subgenera Shigella; 1·1 v. 0·3 %). Salmonella was not detected in faecal samples utilising the pyrosequencing method; however, there was a shift in microbial populations due to clinical status. The clinical symptoms reported herein may be not only due to the Salmonella itself, but also shifts in other gut microbial populations.

scholarly journals Detection of Peanut Allergen Traces with a Real Time PCR Assay - The Challenge to Protect Food-Allergic Consumers

2017 ◽  
Vol 7 (1) ◽  
pp. 32 ◽  
Author(s):  
Dimitra Houhoula ◽  
Stamatios Koussissis ◽  
Vladimiros Lougovois ◽  
John Tsaknis ◽  
Dimitra Kassavita ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Food Products ◽  
Molecular Techniques ◽  
Food Allergens ◽  
Pcr Assay ◽  
Peanut Allergen ◽  
Pcr Method ◽  
Detection And Quantification

The aim of the present study was the implementation of molecular techniques in the detection and quantification of allergic substances of peanut in various kinds of food products, e.g., breakfast cereals, chocolates and biscuits that are frequently related to allergies. In some cases, the presence of peanuts can be due to contamination during production and are not declared on the label. A total of 152 samples were collected from supermarkets and were analysed by a Real Time PCR method. The results indicated that 125 samples (83,3%) were found positive in peanut traces but the most important finding is that from the 84 samples that had no allergen declaration for peanuts, 48 (57,1%) of them were found positive. In conclusion, Real Time PCR can be a very important tool for the rapid detection and quantification of food allergens.

scholarly journals First Report of a Group 16SrII Phytoplasma Associated with Witches'-Broom of Eggplant in Oman

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 360-360 ◽  
Author(s):  
A. M. Al-Subhi ◽  
N. A. Al-Saady ◽  
A. J. Khan ◽  
M. L. Deadman
Keyword(s):  
Nested Pcr ◽  
Solanum Melongena ◽  
Positive Control ◽  
Rrna Gene ◽  
Direct Pcr ◽  
First Report ◽  
Pcr Product ◽  
Pcr Products ◽  
Broom Disease ◽  
Phytoplasma Infection

Eggplant (Solanum melongena L.) belongs to the family Solanaceae and is an important vegetable cash crop grown in most parts of Oman. In February 2010, plants showing phyllody symptoms and proliferation of shoots resembling those caused by phytoplasma infection were observed at Khasab, 500 km north of Muscat. Total genomic DNA was extracted from healthy and two symptomatic plants with a modified (CTAB) buffer method (2) and analyzed by direct and nested PCR with universal phytoplasma 16S rDNA primers P1/P7 and R16F2n/ R16R2, respectively. PCR amplifications from all infected plants yielded an expected product of 1.8 kb with P1/P7 primers and a 1.2-kb fragment with nested PCR, while no products were evident with DNA from healthy plants. Restriction fragment length polymorphism (RFLP) profiles of the 1.2-kb nested PCR products of two eggplant phyllody phytoplasma and five phytoplasma control strains belonging to different groups used as positive control were generated with the restriction endonucleases RsaI, AluI, Tru9I, T-HB8I, and HpaII. The eggplant phytoplasma DNA yielded patterns similar to alfalfa witches'-broom phytoplasma (GenBank Accession No. AF438413) belonging to subgroup 16SrII-D, which has been recorded in Oman (1). The DNA sequence of the 1.8-kb direct PCR product was deposited in GenBank (Accession No. HQ423156). Sequence homology results using BLAST revealed that the eggplant phyllody phytoplasma shared >99% sequence identity with Scaevola witches'-broom phytoplasma (Accession No. AB257291.1), eggplant phyllody phytoplasma (Accession No. FN257482.1), and alfalfa witches'-broom phytoplasma (Accession No. AY169323). The RFLP and BLAST results of 16S rRNA gene sequences confirm that eggplant phyllody phytoplasma is similar to the alfalfa phytoplasma belonging to subgroup 16SrII-D. To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group causing witches'-broom disease on eggplant in Oman. References: (1) A. J. Khan et al. Phytopathology 92:1038, 2002. (2) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA, 81:8014, 1984.

scholarly journals Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tsui-Kang Hsu ◽  
Jung-Sheng Chen ◽  
Hsin-Chi Tsai ◽  
Chi-Wei Tao ◽  
Yu-Yin Yang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
In Silico ◽  
Environmental Samples ◽  
Method Development ◽  
Small Volume ◽  
Detection Efficiency ◽  
Genotyping Method ◽  
Pcr Method

AbstractAcanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

scholarly journals Sex determining of cat embryo and some feline species

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 169-177 ◽  
Author(s):  
Francesca Ciani ◽  
Natascia Cocchia ◽  
Maria Rizzo ◽  
Patrizia Ponzio ◽  
Gennaro Tortora ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Positive Control ◽  
Domestic Cat ◽  
Preimplantation Embryos ◽  
Pcr Analysis ◽  
Domestic Cats ◽  
Hair Samples ◽  
Wild Felids

SummarySex identification in mammalian preimplantation embryos is a technique that is used currently for development of the embryo transfer industry for zootechnical animals and is, therefore, a resource for biodiversity preservation. The aim of the present study was to establish a rapid and reliable method for the sexing of preimplantation embryos in domestic cats. Here we describe the use of nested PCR identify Y chromosome-linked markers when starting from small amounts of DNA and test the method for the purpose of sexing different species of wild felids. To evaluate the efficiency of the primers, PCR analysis were performed first in blood samples of sex-known domestic cats. Cat embryos were produced both in vitro and in vivo and the blastocysts were biopsied. A Magnetic Resin System was used to capture a consistent amount of DNA from embryo biopsy and wild felid hairs. The results from nested PCR applied on cat blood that corresponded to the phenotypical sex. Nested PCR was also applied to 37 embryo biopsies and the final result was: 21 males and 16 females. Furthermore, β-actin was amplified in each sample, as a positive control for DNA presence. Subsequently, nested PCR was performed on blood and hair samples from some wild felines and again the genotyping results and phenotype sex corresponded. The data show that this method is a rapid and repeatable option for sex determination in domestic cat embryos and some wild felids and that a small amount of cells is sufficient to obtain a reliable result. This technique, therefore, affords investigators a new approach that they can insert in the safeguard programmes of felida biodiversity.

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