scholarly journals Comparison of DNA extraction protocols to detect Mycobacterium bovis in bovine tissue by PCR

2016 ◽  
Vol 37 (5Supl2) ◽  
pp. 3709
Author(s):  
Cássia Yumi Ikuta ◽  
Daniella Carvalho Ribeiro Oliveira ◽  
Gisele Oliveira de Souza ◽  
Antonio Francisco de Souza Filho ◽  
José Henrique de Hildebrand Grisi-Filho ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Mycobacterium Bovis ◽  
Gold Standard Method ◽  
Primary Isolation ◽  
Host Interaction ◽  
Detection And Identification ◽  
Specificity And Sensitivity ◽  
Granulomatous Lesions ◽  
Current Scenario ◽  
Suitable Technique

The current scenario of international beef trading has increased the pressure for better and faster diagnosis of bovine tuberculosis. Although traditional culture remains the gold standard method to confirm Mycobacterium bovis infection, it is exceedingly time consuming, and demands viable mycobacteria. Molecular methods overcome the flaws of the bacteriological methods with faster detection and identification. However, mycobacterial features like a complex cell wall and pathogen–host interaction make the molecular detection a challenge. Three protocols for DNA extraction (A, B and C) from bovine tissues were tested to verify the most suitable technique for routine diagnostic assessment of their specificity and sensitivity. Thirty culture-positive and thirty culture-negative granulomatous lesions were included in the trial. From each sample, three tissue suspensions at different dilutions (10-1, 10-2 and 10-3) were prepared and submitted to DNA extraction. PCR procedures targeting IS6110 were performed, employing two volumes of DNA: 5 µL of all three dilutions, and 2.5 µL of the 10-1 dilution. Protocol A was able to detect members of the M. tuberculosis complex in most samples. The sensitivity of the test decreased with increase in tissue-suspension dilution. Although Protocol A presented the highest sensitivity followed by C and B, it showed the lowest specificity, which can be due to a failure in primary isolation caused by the lack of viable organisms or incubation time. Regardless classical bacteriological methods are still recommended by OIE, after evaluating the sensitivity of DNA extraction protocols and PCR procedures, we conclude that the best strategy for M. bovis detection is to follow Protocol A on concentrated tissue suspensions.

scholarly journals Influence of the incubation conditions on culture media to optimize primary isolation of Mycobacterium bovis

2016 ◽  
Vol 37 (5Supl2) ◽  
pp. 3693
Author(s):  
Cássia Yumi Ikuta ◽  
Flávia Morato ◽  
Gisele Oliveira de Souza ◽  
Marcos Bryan Heinemann ◽  
Marcos Amaku ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Bovine Tuberculosis ◽  
Culture Media ◽  
Growth Time ◽  
Solid Culture ◽  
Sodium Pyruvate ◽  
Primary Isolation ◽  
Granulomatous Lesions ◽  
Tuberculosis Diagnosis ◽  
Carbon Dioxide Co2

The isolation of Mycobacterium bovis is critical to a surveillance system for bovine tuberculosis based on detection of lesions in abattoirs. Thus, four solid culture media and three incubation conditions were investigated to elucidate which combination overcomes the others by assessing growth, time to the first appearance of colonies and their number. Ninety-seven samples of granulomatous lesions were submitted to the decontamination procedure by 1-hexadecylpyridinium chloride at 0.75% w/v, and inoculated on two egg-based media, Stonebrink’s (ST) and Löwenstein-Jensen’s with sodium pyruvate (LJp), and two agar-based media, tuberculosis blood agar (B83) and Middlebrook 7H11 medium (7H11). Each medium was incubated at 37°C for 90 days in three incubation conditions: in air, in air containing 10% carbon dioxide (CO2), and in air in slopes closed with burned hydrophobic cotton and subsequently plugged with a cork to create a microaerophilic atmosphere. The colonies appeared faster and in higher number when incubated in air containing 10% CO2 (p < 0.01), independent of media. B83 showed a faster growth and detected more isolates at 30 days of incubation, when compared to ST (0.0178), LJp (p < 0.0001) and 7H11 (p < 0.0001), though there was no difference between B83, ST and LJp at 60 and 90 days of incubation. 7H11 presented the lowest number of isolates (p < 0.0001) and a longer period for the appearance of the first colony (p < 0.001). According to our findings, the concomitant use of ST and B83 media incubated in air containing 10% CO2 increases the isolation of M. bovis in a shorter period of time, which improves bovine tuberculosis diagnosis.

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.

scholarly journals Applications of Nanotechnology in Sensor-Based Detection of Foodborne Pathogens

Sensors ◽  
2020 ◽  
Vol 20 (7) ◽  
pp. 1966 ◽  
Author(s):  
Harsh Kumar ◽  
Kamil Kuča ◽  
Shashi Kant Bhatia ◽  
Kritika Saini ◽  
Ankur Kaushal ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
Pathogen Detection ◽  
Foodborne Pathogen ◽  
Health Issues ◽  
Detection And Identification ◽  
Specificity And Sensitivity ◽  
Food Borne ◽  
Contaminated Food ◽  
Working Principle ◽  
Effective Use

The intake of microbial-contaminated food poses severe health issues due to the outbreaks of stern food-borne diseases. Therefore, there is a need for precise detection and identification of pathogenic microbes and toxins in food to prevent these concerns. Thus, understanding the concept of biosensing has enabled researchers to develop nanobiosensors with different nanomaterials and composites to improve the sensitivity as well as the specificity of pathogen detection. The application of nanomaterials has enabled researchers to use advanced technologies in biosensors for the transfer of signals to enhance their efficiency and sensitivity. Nanomaterials like carbon nanotubes, magnetic and gold, dendrimers, graphene nanomaterials and quantum dots are predominantly used for developing biosensors with improved specificity and sensitivity of detection due to their exclusive chemical, magnetic, mechanical, optical and physical properties. All nanoparticles and new composites used in biosensors need to be classified and categorized for their enhanced performance, quick detection, and unobtrusive and effective use in foodborne analysis. Hence, this review intends to summarize the different sensing methods used in foodborne pathogen detection, their design, working principle and advances in sensing systems.

Detection and identification of poliovirus in environmental samples using nucleic acid hybridization

1990 ◽  
Vol 36 (9) ◽  
pp. 664-669 ◽  
Author(s):  
David R. Preston ◽  
G. Rasul Chaudhry ◽  
Samuel R. Farrah
Keyword(s):  
Tissue Culture ◽  
Nucleic Acid ◽  
Environmental Samples ◽  
Nucleic Acid Hybridization ◽  
Dot Blot ◽  
Polio Virus ◽  
Detection And Identification ◽  
Specificity And Sensitivity ◽  
Hybridization Procedure ◽  
Great Specificity

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage [Formula: see text]. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples. Key words: nucleic acid hybridization, polio virus, water, dot blot.

scholarly journals LAMP Detection and Identification of the Blackleg Pathogen Leptosphaeria biglobosa ‘brassicae’

Plant Disease ◽  
2021 ◽  
Author(s):  
Ran Du ◽  
Yongju Huang ◽  
Jing Zhang ◽  
Long Yang ◽  
Mingde Wu ◽  
...  
Keyword(s):  
Oilseed Rape ◽  
Leptosphaeria Maculans ◽  
Lamp Assay ◽  
Invasive Disease ◽  
Detection And Identification ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Significant Yield ◽  
Pathogen Populations ◽  
Leptosphaeria Biglobosa

Blackleg of oilseed rape is a damaging invasive disease caused by the species complex Leptosphaeria maculans (Lm)/L. biglobosa (Lb), which are composed of at least two and seven phylogenetic subclades, respectively. Generally, Lm is more virulent than Lb, however, under certain conditions, Lb can cause a significant yield loss in oilseed rape. Lb ‘brassicae’ (Lbb) has been found to be the causal agent for blackleg of oilseed rape in China, whereas Lm and Lb ‘canadensis’ (Lbc) were frequently detected in imported seeds of oilseed rape, posing a risk of spread into China. In order to monitor the blackleg-pathogen populations, a diagnostic tool based on loop-mediated isothermal amplification (LAMP) was developed using a 615-bp-long DNA sequence from Lbb that was derived from a randomly amplified polymorphic DNA assay. The LAMP was optimized for temperature and time, and tested for specificity and sensitivity using the DNA extracted from Lbb, Lbc, Lm, and 10 other fungi. The results showed that the optimal temperature and time were 65°C and 40 min, respectively. The LAMP primer set was specific to Lbb and highly sensitive as it detected the Lbb DNA as low as 132 fg per reaction. The LAMP assay was validated using the DNA extracted from mycelia and conidia of a well-characterized Lbb isolate, and its utility was evaluated using the DNA extracted from leaves, stems, pods and seeds of oilseed rape. The LAMP assay developed herein will help for monitoring populations of the blackleg pathogens in China and developing strategies for management of the blackleg disease.

scholarly journals Evaluation of a PCR Primer Based on the Isocitrate Dehydrogenase Gene for Detection of Helicobacter pyloriin Feces

2000 ◽  
Vol 38 (10) ◽  
pp. 3755-3758 ◽  
Author(s):  
Frauke C. Argyros ◽  
Mayurika Ghosh ◽  
Lili Huang ◽  
Naoko Masubuchi ◽  
David R. Cave ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Isocitrate Dehydrogenase ◽  
Specific Primers ◽  
Detection And Identification ◽  
Dna Base ◽  
Dehydrogenase Gene ◽  
H Pylori ◽  
Negative Controls ◽  
Lower Limits ◽  
Different Sources

In order to improve detection and identification ofHelicobacter pylori in highly contaminated samples, we evaluated new specific primers based on the DNA base sequence within the isocitrate dehydrogenase (icd) gene to amplify a 1,200-bp DNA segment. The specificity of the icd primer was tested against DNA derived from various bacteria, including 7Helicobacter species and a panel of 1 gram-variable, 2 gram-positive, and 16 gram-negative bacteria, as well as DNA from houseflies and feces from H. pylori-negative patients. The primers permitted the detection of all clinical H. pyloriisolates tested, but no reactions were observed with negative controls. Several procedures for DNA extraction from feces were evaluated using PCR with icd primers. The lower limits of detection ofH. pylori DNA from two different sources containing the same number of H. pylori organisms, a pure culture and feces spiked with H. pylori, were established for each extraction method tested. The results were 8.0 × 103CFU/ml for cultures of pure H. pylori, and 8.0 × 106 CFU/ml for H. pylori from feces, using the phenol-chloroform method; 8.0 × 102 and 7.0 × 103 CFU/ml, respectively, for a glass matrix and chaotropic solution protocol; 8.0 × 102 and 7.0 × 103 CFU/ml, respectively, for the QIAamp tissue kit; and 5.0 × 102 and 5.0 × 103 CFU/ml, respectively, for the XTRAX DNA extraction kit. We conclude that the use of the icd gene as a primer for PCR represents a specific and sensitive assay for detection of H. pylori in highly contaminated samples.

scholarly journals Circulating 16S RNA in Biofluids: Extracellular Vesicles as Mirrors of Human Microbiome?

2020 ◽  
Vol 21 (23) ◽  
pp. 8959
Author(s):  
Veronica Ricci ◽  
Davide Carcione ◽  
Simone Messina ◽  
Gualtiero I. Colombo ◽  
Yuri D’Alessandra
Keyword(s):  
Extracellular Vesicles ◽  
Human Body ◽  
Human Microbiome ◽  
Microbial Composition ◽  
Gold Standard Method ◽  
Rna Molecules ◽  
Detection And Identification ◽  
16S Rna ◽  
Principal Tool ◽  
Generation Sequencing

The human body is inhabited by around 1013 microbes composing a multicomplex system, termed microbiota, which is strongly involved in the regulation and maintenance of homeostasis. Perturbations in microbiota composition can lead to dysbiosis, which has been associated with several human pathologies. The gold-standard method to explore microbial composition is next-generation sequencing, which involves the analysis of 16S rRNA, an indicator of the presence of specific microorganisms and the principal tool used in bacterial taxonomic classification. Indeed, the development of 16S RNA sequencing allows us to explore microbial composition in several environments and human body districts and fluids, since it has been detected in “germ-free” environments such as blood, plasma, and urine of diseased and healthy subjects. Recently, prokaryotes showed to generate extracellular vesicles, which are known to be responsible for shuttling different intracellular components such as proteins and nucleic acids (including 16S molecules) by protecting their cargo from degradation. These vesicles can be found in several human biofluids and can be exploited as tools for bacterial detection and identification. In this review, we examine the complex link between circulating 16S RNA molecules and bacteria-derived vesicles.

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