scholarly journals Detection of Myocoptes musculinus in Fur Swab and Fecal Samples by Using PCR Analysis

2019 ◽  
Vol 58 (6) ◽  
pp. 796-801
Author(s):  
Mary A Lee ◽  
Zeli Shen ◽  
Hilda R Holcombe ◽  
Zhongming Ge ◽  
Emily G Franklin ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Negative Control ◽  
Qpcr Assay ◽  
Pcr Analysis ◽  
Pcr Assay ◽  
Fecal Samples ◽  
Rna Sequence ◽  
Pcr Assays

Current methods for detecting mites in mouse colonies have limitations in terms of cost, accuracy, and throughput. To address these limitations, we developed PCR assays to detect Myocoptes musculinus in fecal samples. Using a newly generated ribosomal RNA sequence of M. musculinus (MC28S), we developed PCR and qPCR assays capable of detecting M. musculinus mites or eggs ingested during grooming. To determine our ability to detect mites, we tested fur swabs and feces from mouse colonies experimentally infested with M. musculinus and Demodex musculi, 2) Myobia musculi and Radfordia affinis, 3) M. musculinus and M. musculi, and 4) no mites (negative control). The MC28S PCR and qPCR assays positively identified M. musculinus in groups 1 and 3. The MC28S PCR assay detected M. musculinus in 9 of 10 fecal samples from known-positive animals, whereas the qPCR assay correctly identified M. musculinus in all 10 fecal samples. To our knowledge, this report is the first description of PCR-based detection of murine mites in feces. By eliminating the need for pelt examinations, mite detection from fecal samples can facilitate mite detection in sentinel or quarantine programs.

scholarly journals Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting thegroELGene

2012 ◽  
Vol 78 (8) ◽  
pp. 2613-2622 ◽  
Author(s):  
Jana Junick ◽  
Michael Blaut
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Housekeeping Gene ◽  
Cell Number ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Bifidobacterium Adolescentis ◽  
Fecal Samples ◽  
Content Type ◽  
Pcr Assays

ABSTRACTQuantitative real-time PCR assays targeting thegroELgene for the specific enumeration of 12 human fecalBifidobacteriumspecies were developed. The housekeeping genegroEL(HSP60in eukaryotes) was used as a discriminative marker for the differentiation ofBifidobacterium adolescentis,B. angulatum,B. animalis,B. bifidum,B. breve,B. catenulatum,B. dentium,B. gallicum,B. longum,B. pseudocatenulatum,B. pseudolongum, andB. thermophilum. The bifidobacterial chromosome contains a single copy of thegroELgene, allowing the determination of the cell number by quantification of thegroELcopy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a givenBifidobacteriumspecies. Independent of theBifidobacteriumspecies tested, the proportion ofgroELcopies recovered from fecal samples spiked with 5 to 9 log10cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10groELcopies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3Bifidobacteriumspecies andB. longumfrequently detected. The predominant species in infant and adult fecal samples wereB. breveandB. adolescentis, respectively. It was possible to distinguishB. catenulatumandB. pseudocatenulatum. We conclude that thegroELgene is a suitable molecular marker for the specific and accurate quantification of human fecalBifidobacteriumspecies by real-time PCR.

scholarly journals PCR Detection of Group A Bovine Rotaviruses in Feces

1993 ◽  
Vol 5 (4) ◽  
pp. 516-521 ◽  
Author(s):  
Jarasvech Chinsangaram ◽  
Geoffrey Y. Akita ◽  
Anthony E. Castro ◽  
Bennie I. Osburn
Keyword(s):  
Ethidium Bromide ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Pcr Assay ◽  
Fecal Samples ◽  
Bovine Rotavirus ◽  
Viral Particles ◽  
Pcr Inhibitor ◽  
Group A

A polymerase chain reaction (PCR) protocol has been developed for identification of bovine group A rotavirus infection in feces. Primers (20mers) complementary to 3′ ends of double-stranded RNA genome segment 6 of bovine rotavirus NCDV strain were synthesized and used in PCR. Bovine rotavirus RNA from infected cell culture was employed to optimize the PCR protocol. Rotavirus-negative fecal samples were spiked with known quantities of bovine rotavirus, and the sensitivity of the PCR assay was determined. Fecal samples were extracted with phenol and treated to eliminate unidentified PCR inhibitor(s) in feces, and PCR was performed. PCR products were either visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the assay was 6 × 104 viral particles/ml of feces with ethidium bromide-stained agarose gel visualization or 6 × 102 viral particles/ml of feces with chemiluminescent hybridization. The PCR assay was applied to 18 fecal specimens from clinical cases. All 16 clinical samples that were positive for rotavirus by enzyme-linked immunosorbent assay (ELISA) or by ELISA and electron microscopy (EM) were positive by PCR. The 2 samples that were rotavirus negative by ELISA or by ELISA and EM were also negative on PCR analysis.

scholarly journals PARASITOLOGICAL AND MOLECULAR DIAGNOSIS IN EXPERIMENTAL Strongyloides venezuelensis INFECTION

2013 ◽  
Vol 55 (2) ◽  
pp. 141-143 ◽  
Author(s):  
Fabiana Martins Paula ◽  
Renata Barnabé Sitta ◽  
Fernanda Mello Malta ◽  
Maiara Gottardi ◽  
Marcelo Andreetta Corral ◽  
...  
Keyword(s):  
Molecular Diagnosis ◽  
Rattus Norvegicus ◽  
Parasitic Nematode ◽  
Pcr Assay ◽  
Fecal Samples ◽  
Strongyloides Venezuelensis ◽  
Pcr Assays ◽  
Higher Sensitivity

Strongyloides venezuelensis is a parasitic nematode of rats which is frequently used as a model to study human and animal strongyloidiasis. The aim of this study was to evaluate the correlation between parasitological and molecular diagnosis in Strongyloides venezuelensis infection. PCR assays were used to detect S. venezuelensis DNA in fecal samples obtained from experimentally infected Rattus norvegicus. The results showed a higher sensitivity of the PCR assay in detecting the infection compared to parasitological methods.

scholarly journals First report of PCR-based detection of Helicobacter species DNA in Camelus dromedarius in Egypt

2020 ◽  
Vol 13 (9) ◽  
pp. 1898-1901
Author(s):  
Ahmed Youssef ◽  
Ahmed Afifi ◽  
Ayman Hamed ◽  
Mohamed Enany
Keyword(s):  
Ribosomal Rna ◽  
Conventional Polymerase Chain Reaction ◽  
Epidemiological Studies ◽  
Camelus Dromedarius ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Fecal Samples ◽  
First Report ◽  
16S Ribosomal Rna Gene ◽  
Polymerase Chain

Background and Aim: Helicobacter species infections have epidemiological and zoonotic impacts, and different species of Helicobacter have been implicated in infecting humans and animals. The aim of this study was to investigate Helicobacter species infections in Camelus dromedarius. Materials and Methods: Fecal samples were collected from 32 camels from 9 camel farms located at Ismailia Governorate, Egypt. The collected samples were investigated by bacteriological isolation and conventional polymerase chain reaction (PCR) assays targeting the 16S ribosomal RNA gene. Results: Although Helicobacter species could not be isolated from all the examined samples, Helicobacter DNA was detected in 2 (22.22%) of the 9 camel farms. Of the 32 camel fecal samples examined, 4 (12.5%) were positive for Helicobacter species as analyzed by the PCR assay. Conclusion: To the best of our knowledge, this is the first report of PCR-based detection of Helicobacter species infections in C. dromedarius. Further epidemiological studies are required to clarify Helicobacter species infections in camels.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals Genome-Wide Identification and Analysis of the APETALA2 (AP2) Transcription Factor in Dendrobium officinale

2021 ◽  
Vol 22 (10) ◽  
pp. 5221
Author(s):  
Danqi Zeng ◽  
Jaime A. Teixeira da Silva ◽  
Mingze Zhang ◽  
Zhenming Yu ◽  
Can Si ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Flower Development ◽  
Regulatory Elements ◽  
Negative Control ◽  
Dendrobium Officinale ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Luciferase Reporter Gene ◽  
Genome Wide ◽  
Ap2 Transcription Factor

The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, Dendrobium officinale, or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the D. officinale genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all DoAP2 genes contained cis-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of DoAP2-2, DoAP2-5, DoAP2-7, DoAP2-8 and DoAP2-12 genes in protocorm-like bodies (PLBs), while DoAP2-3, DoAP2-4, DoAP2-6, DoAP2-9, DoAP2-10 and DoAP2-11 expression was strong in plantlets. In addition, the expression of some DoAP2 genes was down-regulated during flower development. These results suggest that DoAP2 genes may play roles in plant regeneration and flower development in D. officinale. Four DoAP2 genes (DoAP2-1 from euAP2, DoAP2-2 from euANT, and DoAP2-6 and DoAP2-11 from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of Arabidopsis thaliana mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in Nicotiana benthamiana leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on AP2 genes in D. officinale shed light on the function of AP2 genes in this orchid and other plant species.

scholarly journals Development of a Combined Selection and Enrichment PCR Procedure for Clostridium botulinum Types B, E, and F and Its Use To Determine Prevalence in Fecal Samples from Slaughtered Pigs

2001 ◽  
Vol 67 (10) ◽  
pp. 4781-4788 ◽  
Author(s):  
Maria Dahlenborg ◽  
Elisabeth Borch ◽  
Peter Rådström
Keyword(s):  
Sample Preparation ◽  
Fecal Sample ◽  
Clostridium Botulinum ◽  
Geographical Location ◽  
Type B ◽  
Fecal Samples ◽  
Pcr Assays ◽  
Combined Selection ◽  
Spore Load ◽  
Slaughtered Pigs

ABSTRACT A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymeraserTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70°C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30°C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 × 103 spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.

Phylogenetic relationships of Cryptosporidium determined by ribosomal RNA sequence comparison

1990 ◽  
Vol 20 (2) ◽  
pp. 141-147 ◽  
Author(s):  
Alan M. Johnson ◽  
Robyn Fielke ◽  
Richard Lumb ◽  
Peter R. Baverstock
Keyword(s):  
Ribosomal Rna ◽  
Phylogenetic Relationships ◽  
Sequence Comparison ◽  
Rna Sequence

scholarly journals Use of "EP"(Peroxidase) allele in soybean varietal characterization

2014 ◽  
Vol 36 (4) ◽  
pp. 465-470
Author(s):  
Bárbara Panoff Valário ◽  
Cláudio Cavariani ◽  
José de Barros França-Neto ◽  
Elisa Serra Negra Vieira ◽  
Juliana Pereira Bravo ◽  
...  
Keyword(s):  
Plant Production ◽  
Pcr Analysis ◽  
Agarose Gel Electrophoresis ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Molecular Biology Technique ◽  
Soybean Cultivars ◽  
Colorimetric Test

The aim of this work was to evaluate the use of the molecular biology technique of PCR (Polimerase Chain Reaction) in the characterization of soybean cultivars. The study was performed at the Department of Plant Production, Faculty of Agricultural Sciences/ UNESP and Institute of Bioscience, Botucatu-SP. Fourteen commercial soybean cultivars were used, of which six were selected as positive reaction to peroxidase (BRS 320, BRS 284, BRS 232, BRS 7860RR, BRSMG 760SRR, BRS295RR), four as negative reaction (BRS 326, BRS 8160RR, BRSMG 800A (NutriSoy), BRS Valiosa RR) and four as double reaction (BRSGO 8060, BRS 270RR, FTS Campo Mourão and BRS 239). Thus, the results attained by the traditional biochemical colorimetric test for the 14 cultivars were compared with the conventional PCR assay. For PCR analysis, DNA was extracted from whole seeds and the primers were tested, and subsequently PCR and agarose gel electrophoresis were performed. The combination of primers prx9 + prx10 confirmed the use of the PCR reaction to characterize soybean cultivars considered doubtful by conventional colorimetric text.

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