Specific detection of D- and M-isolates of plum pox virus by immunoenzymatic determination of PCR products

Background and aims: Mucorales are fungi belonging to the category of Zygomycetes, found much in nature. Culture-based methods for clinical samples are often negative, difficult and time-consuming and mainly identify isolates to the genus level, and sometimes only as Mucorales. Therefore, applying fast and accurate diagnosis methods such as molecular approaches seems necessary. This study aims at isolating Mucorales for determination of Rhizopus genus between the isolates using molecular methods. Methods: In this descriptive observational study, a total of 500 samples were collected from air and different surfaces and inoculated on Sabouraud Dextrose Agar supplemented with chloramphenicol. Then, the fungi belonging to Mucorales were identified and their pure culture was provided. DNA extraction was done using extraction kit and the chloroform method. After amplification, the samples belonging to Mucorales were identified by observing 830 bp bands. For enzymatic digestion, enzyme BmgB1 was applied for identification of Rhizopus species by formation of 593 and 235 bp segments. Results: One hundred pure colonies belonging to Mucorales were identified using molecular methods and after enzymatic digestion, 21 isolates were determined as Rhizopus species. The sequencing of PCR products and macroscopic and microscopic studies confirmed the existence of R. stolonifera, R. oryzae and R. caespitosus in the samples. Conclusion: Generally, developing a reliable method for determining Zygomycete species can be a useful tool for better understanding of the epidemiology of mucoromycosis.

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In-house Validation of PCR Based Procedure for Specific Detection of Clostridium Botulinum Types C and D

Bulletin of the Veterinary Institute in Pulawy ◽

10.2478/bvip-2014-0061 ◽

2014 ◽

Vol 58 (3) ◽

pp. 393-398 ◽

Cited By ~ 2

Author(s):

Tomasz Grenda ◽

Elżbieta Kukier ◽

Magdalena Goldsztejn ◽

Krzysztof Kwiatek

Keyword(s):

Clostridium Botulinum ◽

Limit Of Detection ◽

Corn Silage ◽

Laboratory Examination ◽

Specific Detection ◽

Type D ◽

Pcr Products ◽

Type C ◽

Examination Process ◽

Feed Samples

Abstract A PCR-based procedure for detection of C. botulinum C and D in corn silage samples was validated. During the validation, method specificity, sensitivity, and accuracy were determined according to PN - EN ISO 16140:2004. Additionally, the specificity of the validated methods was proved by sequence analysis of PCR products obtained from examination of samples connected with botulism cases in cattle and mallard ducks. Limit of detection was estimated according to the Spearman - Kärber formula and expressed as LOD50. The obtained results showed that a 100% specificity was achieved. The sequencing of PCR products revealed 99% identity with sequences of bont/C and bont/D genes deposited in the GenBank. The sensitivity value ranged from 63.3% for C. botulinum type C to 75% for type D. The accuracy value varied from 72% for type C to 81.3% for type D. LOD50 was estimated at the levels of 0.272 (0. D 188-0395) spore/g for type C and 0.17 (0.1-0.289) spore/g for type D. The described PCR-based procedure enabled detection of C. botulinum C and D at the stage of liquid culture. This makes examination of feed samples possible without isolation process. The presented procedure could support the diagnosis of botulism by faster and specific laboratory examination process.

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Cloth-Based Hybridization Array System for Expanded Identification of the Animal Species Origin of Derived Materials in Feeds

Journal of Food Protection ◽

10.4315/0362-028x-70.12.2900 ◽

2007 ◽

Vol 70 (12) ◽

pp. 2900-2905 ◽

Cited By ~ 4

Author(s):

JOHANNA MURPHY ◽

JENNIFER ARMOUR ◽

BURTON W. BLAIS

Keyword(s):

Mitochondrial Dna ◽

Dna Sequences ◽

Microscopic Examination ◽

Animal Species ◽

Specific Detection ◽

Animal Feeds ◽

Species Origin ◽

Pcr Products ◽

Species Specific ◽

Immunoenzymatic Assay

A cloth-based hybridization array system (CHAS) previously developed for the detection of animal species for which prohibited materials have been specified (cattle, sheep, goat, elk, and deer) has been expanded to include the detection of animal species for which there are no prohibitions (pig and horse) in Canadian and American animal feeds. Animal species were identified by amplification of mitochondrial DNA sequences by PCR and subsequent hybridization of the amplicons with an array of species-specific oligonucleotide capture probes immobilized on a polyester cloth support, followed by an immunoenzymatic assay of the bound PCR products. The CHAS permitted sensitive and specific detection of meat meals from different animal species blended in a grain-based feed and should provide a useful adjunct to microscopic examination for the identification of prohibited materials in animal feeds.

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Specific Detection of the Gene for the Extracellular Neutral Protease of Bacillus cereus by PCR and Blot Hybridization

Applied and Environmental Microbiology ◽

10.1128/aem.65.7.3226-3228.1999 ◽

1999 ◽

Vol 65 (7) ◽

pp. 3226-3228 ◽

Cited By ~ 8

Author(s):

H.-J. Bach ◽

D. Errampalli ◽

K. T. Leung ◽

H. Lee ◽

A. Hartmann ◽

...

Keyword(s):

Bacillus Cereus ◽

Blot Hybridization ◽

Neutral Protease ◽

Gene Probe ◽

Protease Gene ◽

Specific Detection ◽

Pcr Products ◽

Group Members ◽

Restriction Polymorphism

ABSTRACT A pair of primers and a gene probe for the amplification and detection of the Bacillus cereus neutral protease gene (NPRC) were developed. Specificity for the npr genes of theB. cereus group members B. cereus, B. mycoides, and B. thuringiensis was shown. Restriction polymorphism patterns of the PCR products confirmed the presence of the NPRC gene in all three species.

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A new method for rapid detection of Listeria monocytogenes in food

Czech Journal of Food Sciences ◽

10.17221/8319-cjfs ◽

2000 ◽

Vol 18 (No. 3) ◽

pp. 103-109 ◽

Cited By ~ 3

Author(s):

K. Zdeňková ◽

K. Demnerová ◽

G. Jeníková ◽

J. Pazlarová

Keyword(s):

Listeria Monocytogenes ◽

Pcr Primers ◽

Immunomagnetic Separation ◽

Food Samples ◽

Special Importance ◽

Ground Meat ◽

Specific Detection ◽

Gene Encoding ◽

Pcr Method

Listeria monocytogenes represents serious danger for human health. Thus detection of this pathogen in food, which represents its main means of entry into the organism, is a topic of special importance. The original classic methods for the determination of Listeria monocytogenes are in general laborious and time-consuming procedures. In order to address this issue we developed a new rapid method for specific detection of Listeria monocytogenes in food samples. The method consists of three steps: i) enrichment of food microflora (16 h), ii) selective isolation of Listeria sp. exploiting immunomagnetic separation (2–3 h) followed by iii) precise identification of Listeria sp. and Listeria monocytogenes using duplex PCR. PCR primers specific to part of 16S rRNA were used in order to identify the members of Listeria genus. The specific identification of Listeria monocytogenes was accomplished exploiting a pair of primers specific to gene encoding invasion-associated protein – iap (4–5 h). Amplification products, 1003 bp and 593 bp respectively, were separated by electrophoresis and visualized by UV detection. The optimized IMS-PCR method was used to test the presence of Listeria sp. and Listeria monocytogenes in food samples (ground meat, low-fat milk and cheese [olomoucké tvarůžky]). A comparison of the efficiency of the bacteria enrichment step by IMS and centrifugation was also performed. The analysis time including enrichment is less than 24 h. The detection limit for Listeria monocytogenes was found between 101–102 cfu per 25 g of food sample.

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Sex Determination During Inflorescence Bud Differentiation in Monoecious Pistacia chinensis Bunge

Forests ◽

10.3390/f10030202 ◽

2019 ◽

Vol 10 (3) ◽

pp. 202 ◽

Cited By ~ 1

Author(s):

Qian Bai ◽

Chenyi Zhu ◽

Xia Lei ◽

Tao Cao ◽

Shuchai Su ◽

...

Keyword(s):

Sex Determination ◽

Early Stage ◽

Sex Differentiation ◽

Banding Patterns ◽

Vegetative Period ◽

Pcr Products ◽

Polymerase Chain ◽

Sex Determination Mechanisms

Pistacia chinensis Bunge is widely acknowledged to be dioecious, but rare monoecious individuals have been found. However, the origin of monoecism and the sex differentiation of different sex types remain intriguing questions. Here, sex expressions were explored by identification of sex-associated DNA markers, determination of the sex stability after grafting, and histological characterization of inflorescence bud development using anatomical analysis. The results showed that (1) although polymorphisms among individuals existed, the banding patterns of Polymerase Chain Reaction (PCR) products for different sex types on the same monoecious tree were consistent; (2) the sex expressions of grafted trees were not consistent with those of scions, indicating that monoecism probably did not originate from a stable bud mutation; and (3) both males and females underwent a bisexual period, then the stamen primordia in female buds degenerated into the second round tepals, while the pistil primordia in male buds gradually disappeared. During the sex differentiation phase, female buds were spindle-shaped, while the male buds were full teardrop-shaped, and male buds were bigger than female buds. Taken together, no sex-associated DNA marker was found, sex expressions were unstable after grafting, and the alternative sex organs appeared in the early stage of sex differentiation, suggesting that sex determination occurred during floral development instead of the early vegetative period. These results indicated that the sex expressions may be affected by environmental factors, increasing the understanding of sex determination mechanisms in P. chinensis and other species.

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