scholarly journals Identification of aflatoxigenic fungi using polymerase chain reaction-based assay

2014 ◽  
pp. 259-269 ◽  
Author(s):  
Vladislava Soso ◽  
Marija Skrinjar ◽  
Nevena Blagojev ◽  
Slavica Veskovic-Moracanin
Keyword(s):  
Polymerase Chain Reaction ◽  
Regulatory Gene ◽  
Control Point ◽  
Complex Mixture ◽  
Target Sequence ◽  
Chain Reaction ◽  
Isolation And Identification ◽  
Close Link ◽  
Critical Control Point ◽  
Polymerase Chain

As the aflatoxins represent a health-risk for humans because of their proven carcinogenicity, food-borne fungi that produce them as secondary metabolites, mainly Aspergillus flavus and Aspergillus parasiticus, have to be isolated and identified. The best argument for identifying problem fungi is that it indicates control points within the food system as part of a hazard analysis critical control point (HACCP) approach. This assumes there is a close link between fungus and toxin. Conventional methods for isolation and identification of fungi are time consuming and require admirably dedicated taxonomists. Hence, it is imperative to develop methodologies that are relatively rapid, highly specific and as an alternative to the existing methods. The polymerase chain reaction (PCR) facilitates the in vitro amplification of the target sequence. The main advantages of PCR is that organisms need not be cultured, at least not for a long time, prior to their detection, target DNA can be detected even in a complex mixture, no radioactive probes are required, it is rapid, sensitive and highly versatile. The gene afl-2 has been isolated and shown to regulate aflatoxin biosynthesis in A. flavus. Also, the PCR reaction was targeted against aflatoxin synthesis regulatory gene (aflR1) since these genes are nearly identical in A. flavus and A. parasiticus in order to indicate the possibility of detection of both the species with the same PCR system (primers/reaction). [Projekat Ministarstva nauke Republike Srbije, br. III46009] <br><br><font color="red"><b> This article has been retracted. Link to the retraction <u><a href="http://dx.doi.org/10.2298/APT1647265E">10.2298/APT1647265E</a><u></b></font>

Detection of Aspergillus fumigatus by Polymerase Chain Reaction (PCR)

2019 ◽  
Vol 10 (04) ◽  
pp. 655-661
Author(s):  
Zainab H Abood AL-Asadi
Keyword(s):  
Polymerase Chain Reaction ◽  
Aspergillus Fumigatus ◽  
Oligonucleotide Primer ◽  
Rrna Gene ◽  
Fungal Culture ◽  
Chain Reaction ◽  
Isolation And Identification ◽  
Internal Transcribed Spacer Region ◽  
Polymerase Chain ◽  
The Impact

Aspergillosis refers to fungi infections of the respiratory tract caused by Aspergillus species, especially Aspergillus fumigatus. Infection of A. fumigatus was increased in the last few years due to either resistances to antibiotics or the influence of other factors such as other fungal infections. The present study aimed to review the impact of Aspergillus fumigatus in Aspergillosis cases, and study the role of Singleplex PCR for amplification of ITS1, ITS4 of rRNA gene in the detection of fungal isolate. In this study, One hundred sputum samples were collected from patients admitted to the specialize chest and respiratory diseases center / Baghdad who were suffering from respiratory problems. During these studied, molds were isolation and identification based on Conventional method (Direct microscopy by using 10% KOH, and fungal culture was done on Sabouraud Dextrose agar supplemented with chloramphenicol and on Czapek-Dox agar incubated at 37°C and examined for 3-7 days then macroscopic, microscopic examination of the colony by(lactophenol cotton blue stain )and molecular methods by using Polymerase chain reaction (PCR)technique for identification. The 10% KOH examination was positive for 35 cases, while laboratory culturing was positive for 53 cases. Aspergillus sp were isolated from 44(83%) patients; A. fumigatus was isolated in 23 (42. 4%) patients while A. flavus, A. niger, and A. terreus were isolated from 11 (20. 08%), (13. 2%) and 3 (5. 7%) patients respectively, also isolated Penicillium spp. at percentage 1(1. 9%). In this study. The ages of participants ranged from 10-70years with a mean age of 34years, the males were more susceptible to fungal infection, were recorded 35/53 (66. 3), compared to females were 18/53 (33. 96). The infection of fungi was more prevalent in ages 30-40recorded 26(53. 06%) followed by ages 40-50, 13(26. 5), while the lowest infection recorded in the age group 10- 20 years was 2(2. 04%). DNA isolated from twenty-three A. fumigatus isolates was used as a template, and the specific of oligonucleotide primer sequences were used in conventional PCR to detect the presence of internal transcribed spacer region ( ITS) region of the rRNA gene for Aspergillus fumigates. The results of the PCR amplification of the rRNA gene showed that this gene was present in 19 samples out 23 positive samples which isolation with a PCR product size of approximated 385 bp, while 4 samples out 23 positive samples showed negative results for the presence of this gene as indicated by the absence of the PCR products in their relevant lanes. Statistical analysis revealed that the PCR to have a sensitivity of 95. 1% in the detection of Aspergillus fumigatus in Aspergillosis cases. Polymerase chain reaction (PCR) is a rapid, specific, and sensitive method to detect Aspergillus fumigatus in aspergillosis cases of humans.

Poly-β-hydroxybutyrate accumulation in bacterial consortia from different environments

2012 ◽  
Vol 58 (5) ◽  
pp. 660-667 ◽  
Author(s):  
Rahela Carpa ◽  
Anca Butiuc-Keul ◽  
Iulia Lupan ◽  
Lucian Barbu-Tudoran ◽  
Vasile Muntean ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Polymerase Chain Reaction ◽  
Flood Plain ◽  
Soil Samples ◽  
Chain Reaction ◽  
Isolation And Identification ◽  
Stressful Environment ◽  
Transmission Electron ◽  
Polymerase Chain

The aim of the present study was to examine soil samples from various vegetation zones in terms of physicochemical properties, microbial communities, and isolation and identification (by polymerase chain reaction and transmission electron microscopy) of bacteria producing poly-β-hydroxybutyrates (PHBs). Soil samples were analysed originating from zones with heterogeneous environmental conditions from the Romanian Carpathian Mountains (mountain zone with alpine meadow, karstic zone with limestone meadow, hill zone with xerophilous meadow, and flood plain zone with hygrophilic meadow). Different bacterial groups involved in the nitrogen cycle (aerobic mesophilic heterotrophs, ammonifiers, denitrifiers, nitrifiers, and free nitrogen-fixing bacteria from Azotobacter genus) were analysed. Soil biological quality was assessed by the bacterial indicator of soil quality, which varied between 4.3 and 4.7. A colony polymerase chain reaction technique was used for screening PHB producers. With different primers, specific bands were obtained in all the soil samples. Some wild types of Azotobacter species were isolated from the 4 studied sites. Biodegradable polymers of PHB were assessed by negative staining in transmission electron microscopy. The maximum PHB granules density was obtained in the strains isolated from the xerophilous meadow (10–18 granules/cell), which was the most stressful environment from all the studied sites, as the physicochemical and microbiological tests proved.

scholarly journals In SituReverse Transcriptase-Nested Polymerase Chain Reaction to Identify Intracellular Nucleic Acids without the Necessity of Dnase Pretreatment and Hybridisation

2001 ◽  
Vol 22 (3) ◽  
pp. 151-158 ◽  
Author(s):  
Mario Menschikowski ◽  
Margot Vogel ◽  
Rolf Eckey ◽  
Gerd Dinnebier ◽  
Werner Jaross
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acids ◽  
Nested Pcr ◽  
In Situ Hybridisation ◽  
Target Sequence ◽  
Chain Reaction ◽  
Multiple Control ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain

In the present study a protocol of in situ reverse transcriptase‐nested polymerase chain reaction (in situ RT‐nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by “DNA repair mechanisms” and “endogenous priming”, a two‐step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin‐labeled nucleotides and intrinsic primers. (ii) In order to prevent amplifications of genomic DNA nested primer pairs were applied crossing intron sequences. (iii) To minimize the diffusion of PCR products in cells, the extrinsic primers were extended with complementary 5′‐tails. This approach results in the generation of high molecular weight concatamers during PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT‐nested PCR, which in comparison to the method of in situ RT‐PCR‐in situ‐hybridisation is simpler and less time‐consuming, can be used as an alternative approach to identify intracellular nucleic acids.

scholarly journals Evaluation of a Polymerase Chain Reaction-Based Heteroduplex Assay for Detecting the Adulteration of Processed Orange Juice with Mandarin Juice

2006 ◽  
Vol 89 (4) ◽  
pp. 1052-1060 ◽  
Author(s):  
Rachel Mooney ◽  
Louise Chappell ◽  
Angus I Knight
Keyword(s):  
Polymerase Chain Reaction ◽  
Orange Juice ◽  
Polyacrylamide Gel Electrophoresis ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Target Sequence ◽  
Chain Reaction ◽  
Good Repeatability ◽  
Polymerase Chain ◽  
Mandarin Juice

Abstract A polymerase chain reaction (PCR)-based heteroduplex assay was evaluated for the detection of mandarin juice in processed orange juice. PCR amplification of a fragment of the chloroplast trnT-trnL intergenic spacer derived from mixtures of DNA extracted from orange and mandarin juice resulted in heteroduplex formation. The heteroduplex resulted from the co-amplification of a fragment containing an 8 base-pair indel that distinguished mixtures of orange and mandarin juice from orange juice and mandarin juice alone. The heteroduplex assay was evaluated against authentic juices obtained from different citrus species and confirmed that the marker was homogeneous within Citrus. The data obtained demonstrated maternal inheritance of chloroplast type in Citrus sp. and allowed the identification and confirmation of the maternal parentage of unknown and known citrus hybrids. Analysis of the quantitative potential of the PCR and polyacrylamide gel electrophoresis (PAGE) analysis demonstrated good repeatability with a coefficient of variation of 7.5%. Greatest sources of variance in experimental results were attributable to species and varietal differences in the levels of the PCR target. Mandarin juice contained approximately 18% (w/v) less PCR target sequence than did orange juice. The assay was tested in a blind trial using processed juices and correctly identified 20/22 samples with no false-positive results.

scholarly journals Isolation and Identification of Lactic Acid Bacteria from Natural Whey Cultures of Buffalo and Cow Milk

Foods ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 233
Author(s):  
Rosangela Marasco ◽  
Mariagiovanna Gazzillo ◽  
Nicoletta Campolattano ◽  
Margherita Sacco ◽  
Lidia Muscariello
Keyword(s):  
Polymerase Chain Reaction ◽  
Lactic Acid ◽  
Microbial Community ◽  
Lactic Acid Bacteria ◽  
Microbial Diversity ◽  
Strain Level ◽  
Sequencing Analysis ◽  
Chain Reaction ◽  
Isolation And Identification ◽  
Polymerase Chain

In southern Italy, some artisanal farms produce mozzarella and caciocavallo cheeses by using natural whey starter (NWS), whose microbial diversity is responsible for the characteristic flavor and texture of the final product. We studied the microbial community of NWS cultures of cow’s milk (NWSc) for the production of caciocavallo and buffalo’s milk (NWSb) for the production of mozzarella, both from artisanal farms. Bacterial identification at species and strain level was based on an integrative strategy, combining culture-dependent (sequencing of the 16S rDNA, species/subspecies-specific Polymerase Chain Reaction (PCR) and clustering by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) and culture-independent (next-generation sequencing analysis, NGS) approaches. Results obtained with both approaches showed the occurrence of five species of lactic acid bacteria in NWSb (Lactococcus lactis subsp. lactis, Lactobacillus fermentum, Streptococcus thermophilus, Lactobacillus delbrueckii, and Lactobacillus helveticus) and five species in NWSc (Lc. lactis subsp. lactis, Enterococcus faecium, and S. thermophilus, Lb. helveticus, and Lb. delbrueckii), with the last two found only by the NGS analysis. Moreover, RAPD profiles, performed on Lc. lactis subsp. lactis different isolates from both NWSs, showed nine strains in NWSb and seven strains in NWSc, showing a microbial diversity also at strain level. Characterization of the microbiota of natural whey starters aims to collect new starter bacteria to use for tracing microbial community during the production of artisanal cheeses, in order to preserve their quality and authenticity, and to select new Lactic Acid Bacteria (LAB) strains for the production of functional foods.

Detection of Aspergillus fumigatus by Polymerase Chain Reaction(PCR)

2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Zainab H Abood AL-Asadi
Keyword(s):  
Polymerase Chain Reaction ◽  
Aspergillus Fumigatus ◽  
Oligonucleotide Primer ◽  
Rrna Gene ◽  
Fungal Culture ◽  
Chain Reaction ◽  
Isolation And Identification ◽  
Internal Transcribed Spacer Region ◽  
Polymerase Chain ◽  
The Impact

Aspergillosis refers to fungi infections of the respiratory tract caused by Aspergillus species especially Aspergillus fumigatus.Infection of A. fumigatus were increased in last few years , due to either resistances to antibiotics or to influence of other factors such as other fungal infections.The aims of the present study were to review the impact of Aspergillus fumigatus in Aspergillosis cases, and study the role of Singleplex PCR for amplification of ITS1,ITS4 of rRNA gene in the detection of fungal isolate . In this study One hundred sputum samples were collected from patients admitted to the specialize chest and respiratory diseases center / Baghdad who were suffering from respiratory problems. During these studied, molds were isolation and identification based on Conventional method (Direct microscopy by using 10% KOH, and fungal culture was done on Sabouraud Dextrose agar supplemented with chloramphenicol and on Czapek-Dox agar incubated at 37°C and examined for 3-7 days then macroscopic, microscopic examination of the colony by(lactophenol cotton blue stain )and molecular methods by using Polymerase chain reaction (PCR)technique foridentification. The 10% KOH examination was positive for 35 cases while laboratory culturing was positive for 53 cases. Aspergillus sp were isolated from 44(83%) patients; A. fumigatus was isolated in 23 (42.4%) patients while A. flavus, A. niger, and A. terreus were isolated from 11 (20.08%), (13.2%) and 3 (5.7%) patients respectively, also isolated Penicillium spp. at percentage 1(1.9%).. In this study.The ages of participants ranged from 10-70years with a mean age of 34years, the males were more susceptible for fungal infection were recorded 35/53 (66.3), compared to females were 18/53 (33.96).The infection of fungi were more prevalent in ages 30-40recorded 26(53.06%) followed by ages 40-50 ,13(26.5), while the lowest infection recorded in age group 10- 20 years was 2(2.04%). DNA isolated from twenty three A.fumigatus isolates was used as template and the specific of oligonucleotide primer sequences were used in conventional PCR to detect the presence of internal transcribed spacer region ( ITS) region of the rRNA gene for Aspergillus fumigates. The results of the PCR amplification of the rRNA gene showed that, this gene was present in 19 samples out 23 positive samples which isolation with a PCR product size of approximated 385 bp, while 4 samples out 23 positive samples showed negative results for the presence of this gene as indicated by the absence of the PCR products in their relevant lanes.Statistical analysis revealed that the PCR to have a sensitivity of 95.1 % in the detection of Aspergillus fumigatus in Aspergillosis cases.Polymerase chain reaction (PCR) is a rapid, specific and sensitive method to detect Aspergillus fumigatus in aspergillosis cases of human.

scholarly journals Quantitative Real-Time Polymerase Chain Reaction for Bacterial Enumeration and Allelic Discrimination to Differentiate Xanthomonas Strains on Citrus

2005 ◽  
Vol 95 (11) ◽  
pp. 1333-1340 ◽  
Author(s):  
J. Cubero ◽  
J. H. Graham
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Regulatory Protein ◽  
Strain Identification ◽  
Target Sequence ◽  
Chain Reaction ◽  
Allelic Discrimination ◽  
Qrt Pcr ◽  
Plant Samples ◽  
Polymerase Chain

Quantitative real-time polymerase chain reaction (QRT-PCR) was developed for identification and enumeration of bacteria in citrus plant samples infected with Xanthomonas axonopodis pvs. citri and citrumelo, the cause of citrus bacterial canker (CBC) and citrus bacterial spot (CBS), respectively. Three sets of primers based on the pathogenicity gene (pth) in X. axonopodis pv. citri, a ribosomal gene in X. axonopodis pv. citrumelo, and the leucine-responsive regulatory protein (lrp) in both pathovars were combined with TaqMan probes and applied for specific strain detection and quantification. Calibration curves for bacterial abundance in plant samples obtained with the three primer-probe combinations were congruent with colony counts on plates of semiselective medium in most of the cases. However, apparent overestimation of bacterial cells by QRT-PCR indicated the presence of nonculturable or nonviable cells in some samples. In addition to quantification, the lrp primers and probes permitted differentiation by allelic discrimination of Xanthomonas strains infecting citrus tissues. This technique is based on the utilization of two probes that detect a single nucleotide difference in the target sequence between different strains and was validated with a collection of cultured Xanthomonas strains as well as tissue with CBC and CBS lesions. Allelic discrimination is demonstrated to be a more specific and sensitive protocol than previously developed PCR-based methods for strain identification and quantification.

scholarly journals Isolation and identification of Enteropathogenic Escherichia coli (EPEC) in paediatric diarrhoeal patients by detection of bfpA gene by PCR

2013 ◽  
Vol 7 (2) ◽  
pp. 02-05
Author(s):  
Shimu Saha ◽  
Sanya Tahmina Jhora ◽  
Shikha Paul ◽  
Israt Jahan Azmi ◽  
Tarek Mahbub Khan
Keyword(s):  
Polymerase Chain Reaction ◽  
Tertiary Care ◽  
Diarrhoeal Disease ◽  
Care Hospital ◽  
Chain Reaction ◽  
Salmonella Spp ◽  
Isolation And Identification ◽  
Two Samples ◽  
Microbiological Techniques ◽  
Polymerase Chain

This study has been undertaken to investigate the isolation and identification of EPEC strains from paediatric diarrhoeal patients.Total 300 samples were studied.Two hundred and seventy two samples from patients with diarrhoea and  28 samples from control children were collected from two tertiary care hospital. Esch. coli was isolated and identified from all the 300 samples including patient and control using standard microbiological techniques. EPEC strains were identified on the basis of  presence of bundle forming pilus (bfpA) gene. Out of 272 samples from diarrhoeal patient only Esch. coli was isolated from 240 (88.2%) samples. Shigella spp. with Esch. coli were isolated from 27 (10%) specimens and Salmonella spp. with Esch. coli were isolated from 5 (1.8%) samples. Among 272 samples 20 (7.35%) isolates were identified as EPEC on the basis of presence of bfpA gene detected by polymerase chain reaction. EPEC strains were identified from those 240 samples, from which Esch. coli had been isolated only. No EPEC strain was identified  from control children. Rapid and reliable detection of EPEC is required for successful microbiological surveillance and for treatment of EPEC mediated diarrhoeal disease. bfpA gene detection by polymerase chain reaction can be a appropriate method where facilities for polymerase chain reaction are available.DOI: http://dx.doi.org/10.3329/bjmm.v7i2.19324 Bangladesh J Med Microbiol 2013; 07(02): 2-5

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