scholarly journals DEVELOPMENT OF THE REAL-TIME PCR METHODOLOGY FOR TESTING MYCOTOXIGENIC MICROORGANISMS IN GRAPE MARC

2021 ◽  
Vol 28 (3) ◽  
pp. 187-194
Author(s):  
Rodica Sturza ◽  
◽  
Valentin Mitin ◽  
Irina Mitina ◽  
Dan Zgardan ◽  
...  
Keyword(s):  
Real Time ◽  
Ochratoxin A ◽  
Real Time Pcr ◽  
Modern Society ◽  
Fungal Species ◽  
Peptide Sequence ◽  
Wine Production ◽  
The Real ◽  
Grape Marc ◽  
Industrial Waste Management

Agro-industrial waste management is an important problem of modern society as agriculture and food industry are important sources of waste. Wine production generates a considerable amount of winemaking waste (grape marc). Grape marc can be a source of natural dyes, antioxidants and could have various applications, if it is confirmed that it does not contain technogenic contaminants or unwanted microorganisms, for example, producers of mycotoxins. The paper developed the Real -Time Polymerase Chain Reaction (Real-Time PCR) methodology for testing the presence of potentially mycotoxogenic fungal species capable of producing ochratoxin A (OTA), which could be applied before grape marc processing. Based on the non-ribosomal peptide sequence of OTA, involved in ochratoxin biosynthesis, the primers have been developed for the detection of microorganisms potentially capable of producing ochratoxin A.

scholarly journals An improved rapid quantitative detection and identification method for a wide range of fungi

2009 ◽  
Vol 58 (8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Nobutoshi Soeta ◽  
Masanori Terashima ◽  
Mitsukazu Gotoh ◽  
Shuichi Mori ◽  
Kyoko Nishiyama ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Technique ◽  
Fungal Species ◽  
Quantitative Detection ◽  
Its2 Region ◽  
The Real ◽  
Detection And Identification ◽  
Wide Range ◽  
Pcr Products

To develop a rapid and quantitative diagnostic technique for the detection and identification of a wide range of fungi, an improved molecular method based on real-time PCR and the analysis of its products that targets the internal transcribed spacer (ITS) 2 region was established. The real-time PCR could quantitatively and specifically detect the ITS2 region from all 24 tested pathogenic fungal species at between 101 and 107 copies per test without amplification of bacterial or human DNA. The sequences of the primer-binding sites are conserved in the registered sequences of 34 other pathogenic fungal species, suggesting that the PCR would also detect these species. The hyperpolymorphic nature of the ITS2 region between fungal species in terms of length and nucleotide sequence provided valuable information for the determination of species. By labelling the 5′ end of the reverse primer with NED fluorescent dye, the fragment lengths of the real-time PCR products and their 3′-terminal fragments, derived using restriction enzyme ScrFI digestion, were easily evaluated by capillary electrophoresis. Using this analysis, the number and species of fungi present in samples could be estimated. Moreover, sequence analysis of the real-time PCR products could accurately determine species in samples containing a single species. This diagnostic technique can estimate a wide range of fungi from various clinical samples within 1 day and accurately identify them in 2 days. Quantitative results for fungal titre in samples can also provide useful information for understanding the progression of disease and the efficacy of antifungal chemotherapy.

scholarly journals Establishment of a PCR Assay for the Detection and Discrimination of Authentic Cordyceps and Adulterant Species in Food and Herbal Medicines

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 1932 ◽  
Author(s):  
Byeong Moon ◽  
Wook Kim ◽  
Inkyu Park ◽  
Gi-Ho Sung ◽  
Pureum Noh
Keyword(s):  
Dietary Supplements ◽  
Real Time ◽  
Real Time Pcr ◽  
Herbal Medicines ◽  
Fungal Species ◽  
Scar Markers ◽  
Pcr Assay ◽  
Food Ingredients ◽  
The Real ◽  
Species Specific

Accurate detection and differentiation of adulterants in food ingredients and herbal medicines are crucial for the safety and basic quality control of these products. Ophiocordyceps sinensis is described as the only fungal source for the authentic medicinal ingredient used in the herbal medicine “Cordyceps”, and two other fungal species, Cordyceps militaris and Isaria tenuipes, are the authentic fungal sources for food ingredients in Korea. However, substitution of these three species, and adulteration of herbal material and dietary supplements originating from Cordyceps pruinosa or Isaria cicadae, seriously affects the safety and reduces the therapeutic efficacy of these products. Distinguishing between these species based on their morphological features is very difficult, especially in commercially processed products. In this study, we employed DNA barcode-based species-specific sequence characterized amplified region (SCAR) markers to discriminate authentic herbal Cordyceps medicines and Cordyceps-derived dietary supplements from related but inauthentic species. The reliable authentication tool exploited the internal transcribed spacer (ITS) region of a nuclear ribosomal RNA gene (nrDNA). We used comparative nrDNA-ITS sequence analysis of the five fungal species to design two sets of SCAR markers. Furthermore, we used a set of species-specific SCAR markers to establish a real-time polymerase chain reaction (PCR) assay for the detection of species, contamination, and degree of adulteration. We confirmed the discriminability and reproducibility of the SCAR marker analysis and the real-time PCR assay using commercially processed food ingredients and herbal medicines. The developed SCAR markers may be used to efficiently differentiate authentic material from their related adulterants on a species level. The ITS-based SCAR markers and the real-time PCR assay constitute a useful genetic tool for preventing the adulteration of Cordyceps and Cordyceps-related dietary supplements.

scholarly journals A Real-Time, Quantitative PCR Seed Assay for Botrytis spp. that Cause Neck Rot of Onion

Plant Disease ◽  
2007 ◽  
Vol 91 (5) ◽  
pp. 599-608 ◽  
Author(s):  
Martin I. Chilvers ◽  
Lindsey J. du Toit ◽  
Hajime Akamatsu ◽  
Tobin L. Peever
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Intergenic Spacer ◽  
Fungal Species ◽  
Pcr Assay ◽  
The Real ◽  
Real Time Quantitative Pcr ◽  
Onion Seed ◽  
Pure Cultures ◽  
Fluorescent Polymerase Chain Reaction

A real-time fluorescent polymerase chain reaction (PCR) assay was developed using SYBR Green chemistry to quantify the Botrytis spp. associated with onion (Allium cepa) seed that are also able to induce neck rot of onion bulbs, i.e., B. aclada, B. allii, and B. byssoidea. The nuclear ribosomal intergenic spacer (IGS) regions of target and nontarget Botrytis spp. were sequenced, aligned, and used to design a primer pair specific to B. aclada, B. allii, and B. byssoidea. Primers and amplification parameters were optimized to avoid amplifying the related species B. cinerea, B. porri, and B. squamosa, as well as Sclerotinia sclerotiorum and isolates of 15 other fungal species commonly found associated with onion seed. The primers reliably detected 10 fg of genomic DNA per PCR reaction extracted from pure cultures of B. aclada and B. allii. Conventional assays of surface-disinfested and nondisinfested seed on an agar medium were used to determine the incidence of neck rot Botrytis spp. associated with each of 23 commercial onion seed lots, and the real-time PCR assay was used to determine the quantity of DNA of neck rot Botrytis spp. in each seed lot. A linear relationship could not be found between the incidence of seed infected with the neck rot Botrytis spp. using the conventional agar seed assays and the quantity of DNA of the neck rot Botrytis spp. detected by the real-time PCR assay. However, the real-time PCR assay appeared to be more sensitive than the conventional agar assay, allowing detection of neck rot Botrytis spp. in 5 of the 23 seed lots that tested negative using the conventional agar seed assay.

APPLICATION OF REAL-TIME PRC TECHNIQUE AND REVERSE DOT-BLOT FOR DETECTION THE HIGH RISK TYPES OF HUMAN PAPILLOMAVIRUS CAUSING CERVICAL CANCER

2017 ◽  
pp. 99-103
Author(s):  
Van Bao Thang Phan ◽  
Hoang Bach Nguyen ◽  
Van Thanh Nguyen ◽  
Thi Nhu Hoa Tran ◽  
Viet Quynh Tram Ngo
Keyword(s):  
Cervical Cancer ◽  
High Risk ◽  
Real Time ◽  
Real Time Pcr ◽  
Dot Blot ◽  
The Real ◽  
Dot Blot Assay ◽  
Hpv Types ◽  
High Risk Hpv ◽  
Reverse Dot Blot

Introduction: Infection with HPV is the main cause of cervical cancer. Determining HPV infection and the types of HPV plays an important role in diagnosis, treatment and prognosis of cervicitis/cervical cancer. Aims: Determining proportion of high-risk HPV types and the occurrence of coinfection with multiple HPV types. Methods: 177 women with cervicitis or abnormal Pap smear result were enrolled in the study. Performing the real-time PCR for detecting HPV and the reverse DOT-BLOT assay for determining type of HPV in cases of positive PCR. Results: 7 types of high-risk HPV was dectected, the majority of these types were HPV type 18 (74.6%) and HPV type 16 (37.6%); the proportion of infection with only one type of HPV was 30.4% and coinfection with multiple HPV types was higher (69.6%), the coinfected cases with 2 and 3 types were dominated (32.2% and 20.3%, respectively) and the coinfected cases with 4 and 5 types were rare. Conclusion: Use of the real-time PCR and reverse DOT-BLOT assay can determine the high-risk HPV types and the occurrence of coinfection with multiple HPV types. Key words: HPV type, Reverse DOT-BLOT, real-time PCR,PCR, cervical cancer

scholarly journals Real-Time PCR as an Alternative Technique for Detection of Dermatophytes in Cattle Herds

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1662
Author(s):  
Dominik Łagowski ◽  
Sebastian Gnat ◽  
Aneta Nowakiewicz ◽  
Aleksandra Trościańczyk
Keyword(s):  
Light Microscopy ◽  
Real Time ◽  
Real Time Pcr ◽  
Direct Analysis ◽  
Carrier Status ◽  
The Real ◽  
Detection Techniques ◽  
Direct Light ◽  
Microscopy Analysis ◽  
Cattle Herds

Dermatophytes are filamentous fungi with the ability to digest and grow on keratinized substrates. The ongoing improvements in fungal detection techniques give new scope for clinical implementations in laboratories and veterinary clinics, including the monitoring of the disease and carrier status. The technologically advanced methods for dermatophyte detection include molecular methods based on PCR. In this context, the aim of this study was to carry out tests on the occurrence of dermatophytes in cattle herds using qPCR methods and a comparative analysis with conventional methods. Each sample collected from ringworm cases and from asymptomatic cattle was divided into three parts and subjected to the real-time PCR technique, direct light microscopy analysis, and culture-based methods. The use of the real-time PCR technique with pan-dermatophyte primers detected the presence of dermatophytes in the sample with a 10.84% (45% vs. 34.17%) higher efficiency than direct analysis with light microscopy. Moreover, a dermatophyte culture was obtained from all samples with a positive qPCR result. In conclusion, it seems that this method can be used with success to detect dermatophytes and monitor cowsheds in ringworm cases and carriers in cattle.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

scholarly journals Performance Evaluation of the Real-Q Cytomegalovirus (CMV) Quantification Kit Using Two Real-Time PCR Systems for Quantifying CMV DNA in Whole Blood

2016 ◽  
Vol 36 (6) ◽  
pp. 603-606 ◽  
Author(s):  
Jong Eun Park ◽  
Ji-Youn Kim ◽  
Sun Ae Yun ◽  
Myoung-Keun Lee ◽  
Hee Jae Huh ◽  
...  
Keyword(s):  
Performance Evaluation ◽  
Real Time ◽  
Real Time Pcr ◽  
Whole Blood ◽  
The Real

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

scholarly journals Touchable 3D hierarchically structured polyaniline nanoweb for capture and detection of pathogenic bacteria

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Kyung Hoon Kim ◽  
MinHo Yang ◽  
Younseong Song ◽  
Chi Hyun Kim ◽  
Young Mee Jung ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Bacterial Pathogens ◽  
Physical Contact ◽  
Structural Deformation ◽  
Pcr Analysis ◽  
Mechanical Resistance ◽  
Fluorescence Signal ◽  
The Real

AbstractA bacteria-capturing platform is a critical function of accurate, quantitative, and sensitive identification of bacterial pathogens for potential usage in the detection of foodborne diseases. Despite the development of various nanostructures and their surface chemical modification strategies, relative to the principal physical contact propagation of bacterial infections, mechanically robust and nanostructured platforms that are available to capture bacteria remain a significant problem. Here, a three-dimensional (3D) hierarchically structured polyaniline nanoweb film is developed for the efficient capture of bacterial pathogens by hand-touching. This unique nanostructure ensures sufficient mechanical resistance when exposed to compression and shear forces and facilitates the 3D interfacial interactions between bacterial extracellular organelles and polyaniline surfaces. The bacterial pathogens (Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus) are efficiently captured through finger-touching, as verified by the polymerase chain reaction (PCR) analysis. Moreover, the real-time PCR results of finger-touched cells on a 3D nanoweb film show a highly sensitive detection of bacteria, which is similar to those of the real-time PCR using cultured cells without the capturing step without any interfering of fluorescence signal and structural deformation during thermal cycling. Graphic Abstract

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