scholarly journals Rapid and Simple Detection of Trichosporon asahii by Optimized Colony PCR

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Dequan Zhang ◽  
Xuelian Lu ◽  
Yong Liao ◽  
Zhikuan Xia ◽  
Zhuoying Peng ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Intergenic Spacer ◽  
Glass Beads ◽  
Clinical Samples ◽  
Trichosporon Asahii ◽  
Colony Pcr ◽  
Extraction And Purification ◽  
Simple Detection ◽  
Mock Infection ◽  
Species Specific

Trichosporon asahii is the major pathogen causing invasive trichosporonosis. Conventional methods of its detection are time-consuming or costly and often require complex DNA extraction and purification steps, which hinders rapid clinical diagnosis. In this study, we evaluated colony PCR, which directly uses colonies or trace clinical samples as the template for amplification, for rapid detection of T. asahii infection. Four methods, namely, direct colony, freeze-thaw, glass beads, and enzymolysis, were compared to select the best DNA extraction strategy. We subsequently designed and screened species-specific primers targeting the intergenic spacer 1 (IGS1) of the ribosomal DNA of T. asahii and used them to detect mock infection clinical samples. The species-specific colony PCR based on glass beads proved advantageous, with short procedure time (154.8 ± 0.6 min), good sensitivity (detection limit, 102 CFU/mL), and specificity for T. asahii, indicating that this method can be used for the rapid and simple identification of clinical samples of T. asahii infection.

scholarly journals Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections

2020 ◽  
Vol 6 (4) ◽  
pp. 308
Author(s):  
Joana Carvalho-Pereira ◽  
Filipa Fernandes ◽  
Ricardo Araújo ◽  
Jan Springer ◽  
Juergen Loeffler ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Invasive Fungal Infections ◽  
Clinical Samples ◽  
Correct Identification ◽  
Colony Pcr ◽  
Pcr Multiplex ◽  
Pathogen Dna ◽  
Species Specific

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.

scholarly journals Identification of Porphyromonas gingivalis Strains by Heteroduplex Analysis and Detection of Multiple Strains

1999 ◽  
Vol 37 (12) ◽  
pp. 3906-3911 ◽  
Author(s):  
Eugene J. Leys ◽  
James H. Smith ◽  
Sharon R. Lyons ◽  
Ann L. Griffen
Keyword(s):  
Porphyromonas Gingivalis ◽  
Dna Sequences ◽  
Allelic Variation ◽  
Intergenic Spacer ◽  
Heteroduplex Analysis ◽  
Clinical Samples ◽  
Specific Primers ◽  
Intergenic Spacer Region ◽  
Species Specific ◽  
Multiple Strains

Heteroduplex analysis has been used extensively to identify allelic variation among mammalian genes. It provides a rapid and reliable method for determining and cataloging minor differences between two closely related DNA sequences. We have adapted this technique to distinguish among strains or clonal types of Porphyromonas gingivalis. The ribosomal intergenic spacer region (ISR) was amplified directly from a subgingival plaque sample by PCR with species-specific primers, avoiding the need for culturing the bacteria. The PCR products were then directly compared by heteroduplex analysis with known strains of P. gingivalis for identification. We identified 22 distinct but closely related heteroduplex types ofP. gingivalis in 1,183 clinical samples. Multiple strains were found in 34% of the samples in which P. gingivaliswas detected. Heteroduplex types were identified from these multistrain samples without separating them by culturing or molecular cloning. PCR with species-specific primers and heteroduplex analysis makes it possible to reliably and sensitively detect and identify strains ofP. gingivalis in large numbers of samples.

scholarly journals Strain Typing of Trichosporon asahii Clinical Isolates by Random Amplification of Polymorphic DNA (RAPD) Analysis

2021 ◽  
Author(s):  
Thayanidhi Premamalini ◽  
Vijayaraman Rajyoganandh ◽  
Ramaraj Vijayakumar ◽  
Hemanth Veena ◽  
Anupma Jyoti Kindo ◽  
...  
Keyword(s):  
Genetic Relatedness ◽  
Rapd Analysis ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Strain Typing ◽  
Trichosporon Asahii ◽  
Pcr Fingerprinting ◽  
Specific Pcr ◽  
Rapd Primer ◽  
Random Amplification

Abstract Objective The aim of this study was to identify and isolate Trichosporon asahii (T. asahii) from clinical samples and to assess the genetic relatedness of the most frequently isolated strains of T. asahii using random amplification of polymorphic DNA (RAPD) primers GAC-1 and M13. Methods All the clinical samples that grew Trichosporon species, identified and confirmed by polymerase chain reaction (PCR) using Trichosporon genus-specific primers, were considered for the study. Confirmation of the species T. asahii was carried out by T. asahii-specific PCR. Fingerprinting of the most frequently isolated T. asahii isolates was carried out by RAPD using random primers GAC-1 and M13. Results Among the 72 clinical isolates of Trichosporon sp. confirmed by Trichosporon-specific PCR, 65 were found to be T. asahii as identified by T. asahii-specific PCR. Fingerprinting of the 65 isolates confirmed as T. asahii using GAC-1 RAPD primer yielded 11 different patterns, whereas that of M13 primer produced only 5 patterns. The pattern I was found to be the most predominant type (29.2%) followed by pattern III (16.9%) by GAC-1 primer. Conclusions This study being the first of its kind in India on strain typing of T. asahii isolates by adopting RAPD analysis throws light on genetic diversity among the T. asahii isolates from clinical samples. Fingerprinting by RAPD primer GAC-1 identified more heterogeneity among the T. asahii isolates than M13.

Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension)

The Analyst ◽  
2012 ◽  
Vol 137 (17) ◽  
pp. 4023 ◽  
Author(s):  
Lindsay N. Strotman ◽  
Guangyun Lin ◽  
Scott M. Berry ◽  
Eric A. Johnson ◽  
David J. Beebe
Keyword(s):  
Surface Tension ◽  
Dna Extraction ◽  
Extraction And Purification ◽  
Food Matrices

scholarly journals A Review of Murine Cytomegalovirus as a Model for Human Cytomegalovirus Disease—Do Mice Lie?

2020 ◽  
Vol 22 (1) ◽  
pp. 214
Author(s):  
Michelle A. Fisher ◽  
Megan L. Lloyd
Keyword(s):  
Human Cytomegalovirus ◽  
Murine Cytomegalovirus ◽  
Clinical Samples ◽  
Mouse Strains ◽  
Cytomegalovirus Disease ◽  
Wild Mice ◽  
Congenital Cytomegalovirus ◽  
Non Invasive ◽  
Species Specific ◽  
Disseminated Disease

Since murine cytomegalovirus (MCMV) was first described in 1954, it has been used to model human cytomegalovirus (HCMV) diseases. MCMV is a natural pathogen of mice that is present in wild mice populations and has been associated with diseases such as myocarditis. The species-specific nature of HCMV restricts most research to cell culture-based studies or to the investigation of non-invasive clinical samples, which may not be ideal for the study of disseminated disease. Initial MCMV research used a salivary gland-propagated virus administered via different routes of inoculation into a variety of mouse strains. This revealed that the genetic background of the laboratory mice affected the severity of disease and altered the extent of subsequent pathology. The advent of genetically modified mice and viruses has allowed new aspects of disease to be modeled and the opportunistic nature of HCMV infection to be confirmed. This review describes the different ways that MCMV has been used to model HCMV diseases and explores the continuing difficulty faced by researchers attempting to model HCMV congenital cytomegalovirus disease using the mouse model.

scholarly journals Optimizing DNA extraction methods for Nanopore sequencing of Neisseria gonorrhoeae direct from urine samples

2019 ◽  
Author(s):  
Teresa L. Street ◽  
Leanne Barker ◽  
Nicholas D. Sanderson ◽  
James Kavanagh ◽  
Sarah Hoosdally ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Reference Genome ◽  
Extraction Methods ◽  
Urine Samples ◽  
Clinical Samples ◽  
Nanopore Sequencing ◽  
Human Dna ◽  
Whole Genomes ◽  
Dna Extraction Methods ◽  
Multiple Antibiotics

AbstractBackgroundEmpirical gonorrhoea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance.MethodsWe investigated if Nanopore sequencing can detect sufficient N. gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae spiked urine samples and samples from gonorrhoea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced whilst minimizing contaminating host DNA.ResultsIn simulated infections the Qiagen UCP Pathogen Mini kit provided the highest ratio N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections, but decreased yields in clinical samples. In ten urine samples from men with symptomatic urethral gonorrhoea, ≥87% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥92% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR Media tubes and from urethral swabs, and in the presence of simulated Chlamydia co-infection.ConclusionUsing Nanopore sequencing of urine samples from men with urethral gonorrhoea sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.

scholarly journals A Powerful DNA Extraction Method and PCR for Detection of Microsporidia in Clinical Stool Specimens

1999 ◽  
Vol 6 (2) ◽  
pp. 243-246 ◽  
Author(s):  
Andreas Müller ◽  
Kerstin Stellermann ◽  
Pia Hartmann ◽  
Matthias Schrappe ◽  
Gerd Fätkenheuer ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Dna Extraction ◽  
Extraction Method ◽  
Clinical Samples ◽  
Species Differentiation ◽  
Specific Method ◽  
Dna Extraction Method ◽  
Specificity And Sensitivity ◽  
Stool Specimens ◽  
Intestinal Microsporidiosis

ABSTRACT The diagnosis of intestinal microsporidiosis has traditionally depended on direct visualization of the parasite in stool specimens or intestinal biopsy samples by light and/or electron microscopy. Limited information about the specificity and sensitivity of PCR for the detection microsporidia in clinical stool specimens is available. To establish a sensitive and specific method for the detection of microsporidia in clinical samples, we studied clinical stool specimens of 104 randomly selected human immunodeficiency virus-infected patients with diarrhea to compare light microscopy and PCR. Fluorochrome Uvitex 2B staining was used for light microscopy. To raise the sensitivity of PCR, we used a powerful and fast DNA extraction method including stool sedimentation, glass bead disruption, and proteinase K and chitinase digestion. PCR was performed with primer pairs V1-PMP2, V1-EB450, and V1-SI500, and the nature of the PCR products was confirmed by Southern blot hybridization. Microsporidiosis was diagnosed by light microscopy in eight patients. Ten patients tested positive for microsporidiosis by PCR. Enterocytozoon bieneusi was found in seven cases, andEncephalitozoon intestinalis was found in four cases. In one case a double infection with E. bieneusi and E. intestinalis was diagnosed by PCR, whereas light microscopy showed only E. bieneusi infection. PCR testing of stool specimens is useful for diagnosis and species differentiation of intestinal microsporidiosis in HIV patients.

A comparison of DNA extraction and purification methods to detect Escherichia coli O157:H7 in cattle manure

2003 ◽  
Vol 54 (2) ◽  
pp. 165-175 ◽  
Author(s):  
Teegan Trochimchuk ◽  
John Fotheringham ◽  
Edward Topp ◽  
Heidi Schraft ◽  
Kam Tin Leung
Keyword(s):  
Escherichia Coli ◽  
Dna Extraction ◽  
Cattle Manure ◽  
Escherichia Coli O157 ◽  
Extraction And Purification ◽  
Purification Methods

Development of an efficient PCR-based diagnosis protocol for the identification of the pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

Nematology ◽  
2004 ◽  
Vol 6 (2) ◽  
pp. 279-285 ◽  
Author(s):  
Jae Soon Kang ◽  
Kwang Sik Choi ◽  
Sang Chul Shin ◽  
Il Sung Moon ◽  
Sang Gil Lee ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Nematode Species ◽  
Bursaphelenchus Xylophilus ◽  
5S Rrna ◽  
Rrna Gene ◽  
Pinewood Nematode ◽  
Pine Wood ◽  
5S Rrna Gene ◽  
Primer Sets ◽  
Species Specific

Abstract Pine wood wilt disease caused by the pine wood nematode, Bursaphelenchus xylophilus , has been a serious problem in the southern regions of Korea. Efficient diagnosis of B. xylophilus from infected pine wood specimens is critical for the management of this pest. Traditional microscopic examination often results in an erroneous identification because a closely related non-pathogenic species, B. mucronatus, has a great degree of morphological similarity to B. xylophilus. In an attempt to search for reliable molecular markers for the discrimination of these species, we have cloned the 5S rRNA genomic DNA fragments containing both coding and intergenic spacer (IGS) regions from B. xylophilus and B. mucronatus through a homology-probing PCR strategy. Sequence analyses revealed that coding sequences of the 5S rRNA gene from the two species are almost identical (98.3% homology) but that the IGS sequences differ substantially between the species. Based on the IGS sequence differences (69.7% homology), we designed species-specific primer sets and developed a PCR-based diagnosis protocol for the identification and discrimination of the two nematode species on a molecular basis.

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