A PCR Method That Can Be Further Developed into PCR-RFLP Assay for Eight Animal Species Identification

There are many PCR-based methods for animal species identification; however, their detection numbers are limited or could not identify unknown species. We set out to solve this problem by developing a universal primer PCR assay for simultaneous identification of eight animal species, including goat, sheep, deer, buffalo, cattle, yak, pig, and camel. In this assay, the variable lengths of mitochondrial DNA were amplified using a pair of universal primers. PCR amplifications yielded 760 bp, 737 bp, 537 bp, 486 bp, 481 bp, 464 bp, 429 bp, and 359 bp length fragments for goat, sheep, deer, buffalo, cattle, yak, pig, and camel, respectively. This primer pair had no cross-reaction with other common domestic animals and fish. The limit of detection varied from 0.01 to 0.05 ng of genomic DNA for eight animal species in a 20 µl PCR mixture. Each PCR product could be further digested into fragments with variable sizes and qualitative analysis by SspI restriction enzyme. This developed PCR-RFLP assay was sufficient to distinguish all targeted species. Compared with the previous published related methods, this approach is simple, with high throughput, fast processing rates, and more cost-effective for routine identification of meat in foodstuffs.

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Fast and reliable PCR/sequencing/RFLP assay for identification of fungi in onychomycoses

Journal of Medical Microbiology ◽

10.1099/jmm.0.46723-0 ◽

2006 ◽

Vol 55 (9) ◽

pp. 1211-1216 ◽

Cited By ~ 41

Author(s):

Michel Monod ◽

Olympia Bontems ◽

Christophe Zaugg ◽

Barbara Léchenne ◽

Marina Fratti ◽

...

Keyword(s):

Rflp Analysis ◽

Fungal Species ◽

Universal Primers ◽

Cure Rate ◽

Routine Identification ◽

Identification Of Fungi ◽

Pcr Products ◽

Nail Sample ◽

Fungal Dna ◽

Rflp Assay

Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from abnormal nails. To investigate whether these fungi are the aetiological agents of infection or simply transient contaminants, a PCR/sequencing/RFLP assay was developed for direct and routine identification of the infecting fungi in onychomycosis. Fungal DNA was readily extracted using a commercial kit after dissolving nail fragments in a Na2S solution. Amplification of part of the 28S rDNA by PCR was performed with universal primers and the fungal species were identified by sequencing. The PCR/sequencing results were comparable with microbiological identification from the same nail sample. In addition to dermatophytes, Fusarium spp. and other less frequently isolated non-dermatophyte fungi were identified as single fungal agents in onychomycosis. Moreover, mixed infections were clearly demonstrated in 10 % of cases by RFLP analysis of PCR products. Identification of infectious agents could be obtained in 2 days, whilst results from fungal cultures take 1–3 weeks. Rapid and reliable molecular identification of the infectious fungus expedites the choice of appropriate antifungal therapy, thereby improving the cure rate of onychomycosis.

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Identification of Eutypa lata by PCR-RFLP

Plant Disease ◽

10.1094/pdis.2004.88.9.925 ◽

2004 ◽

Vol 88 (9) ◽

pp. 925-929 ◽

Cited By ~ 26

Author(s):

P. E. Rolshausen ◽

F. Trouillas ◽

W. D. Gubler

Keyword(s):

Universal Primers ◽

Phaeomoniella Chlamydospora ◽

Phomopsis Viticola ◽

Eutypa Lata ◽

Incorrect Diagnosis ◽

Pcr Rflp ◽

Pcr Method ◽

Polymerase Chain ◽

Rflp Assay

Eutypa lata is a vascular canker pathogen of woody plants commonly diagnosed by isolating the pathogen from infected tissue. Related fungi from the same family, the Diatrypaceae, also have been found in association with grapevine in Californian vineyards. An in situ polymerase chain reaction (PCR) method has been developed for detection of E. lata in infected wood tissue. However, our results indicate that this method also would amplify other Diatrypaceous fungi, which could potentially lead to an incorrect diagnosis. Therefore, we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay. The internal transcribed spacer (ITS)1/5.8S/ITS2 ribosomal DNA region was amplified by PCR using universal primers, and RFLP patterns were determined after digestion with AluI. The restriction profiles obtained served to distinguish E. lata from wood trunk pathogens of grapevine (Phomopsis viticola, Botryodiplodia sp., Phaeoacremonium aleophilum, and Phaeomoniella chlamydospora), Diatrypaceous fungi (Diatrype sp., Diatrypella sp., Eutypella vitis, and Eutypa leptoplaca), and Cryptovalsa sp. found on dead wood of grapevine, and other Eutypa spp. (E.petrakii var. hederae, E. astroidea, E. crustata, and E. lejoplaca), with the exception of E. armeniacae, which we regard as a synonym for E. lata, and E. laevata, which has not been found on grapevine.

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8.Animal Species Identification through High Resolution Melting Real Time PCR (HRM) of the Mitochondrial 16S rRNA Gene

Annals of Animal Science ◽

10.1515/aoas-2015-0074 ◽

2016 ◽

Vol 16 (2) ◽

pp. 415-424 ◽

Cited By ~ 3

Author(s):

Pitchayanipa Klomtong ◽

Yupin Phasuk ◽

Monchai Duangjinda

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Species Identification ◽

Animal Species ◽

Restriction Enzymes ◽

Rrna Gene ◽

16S Rrna Gene Analysis ◽

Universal Primer ◽

Pcr Product ◽

Mitochondrial 16S Rrna Gene

Abstract Animal species identification has received growing attention, regarding genetic diversity and food traceability. The objective of this study is to apply a universal primer of part of the mitochondrial 16S rRNA gene analysis using the PCR-RFLP and HRM methods for identification of species origin in cattle, chicken, horse, sheep, pig, buffalo, and goat. PCR product size was 512 bp. The PCR product of 16S rRNA was digested with two restriction enzymes (BclI and MseI); sufficient to easily generate analyzable species-specific restriction profiles that could distinguish the unambiguity of all targeted species. The HRM method successfully identified all species by shape of melting temperature, and proved to be of higher resolution, and a more cost effective, alternative method compared with other identification techniques.

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Universal Fragment of mtDNA for Cervidae and Other Ungulate Species Identification

10.21203/rs.3.rs-117242/v1 ◽

2020 ◽

Author(s):

Ewa Filip ◽

Tomasz Strzała ◽

Edyta Stępień ◽

Klaudia Pizoń

Keyword(s):

Species Identification ◽

Cervus Elaphus ◽

Animal Species ◽

Capreolus Capreolus ◽

Identification Procedure ◽

Free Living ◽

Degraded Samples ◽

Mitochondrial Cytochrome B ◽

Routine Identification

Abstract ObjectiveThe abundance of literature and many studies aimed at the identification of free-living animal species has helped to identify specific nucleotide marker sequences. In many cases, the best marker to distinguish species is the mitochondrial genome (mtDNA) or one of its fragments. In molecular analyses of Cervidae, biological material such as blood or muscle is easily obtained, as nearly all of these species are gaming animals.ResultsIn our research, we present the case study of successful species identification based on degraded samples of bone, with the use of short mtDNA fragments. We obtained a partial sequence of the mitochondrial cytochrome b (Cytb) gene for Capreolus capreolus, Dama dama, and Cervus elaphus, that can be used for species affiliation. The proposed methodology is helpful as a routine identification procedure for a variety of tissue sources, even in cases where the samples are degraded. The new sequences have been deposited in GenBank, enriching the existing Cervidae mtDNA base.

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PCR-RFLP Detection od PAI-2 Gene Variants: Prevelence in Ethnic Groups and Disease Relationship in patients Undergoing corony Angiography

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656084 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0955-0958 ◽

Cited By ~ 8

Author(s):

Carole A Foy ◽

Peter J Grant

Keyword(s):

Gene Variants ◽

Genotype Distribution ◽

Pima Indians ◽

Significant Difference ◽

Pcr Rflp ◽

History Of ◽

Caucasian Patients ◽

Clinical Conditions ◽

Rflp Assay ◽

Coronary Atheroma

SummaryPAI-2 is a fibrinolytic inhibitor produced predominantly by monocytes. Most PAI-2 is intracellular making study in clinical conditions difficult. Abnormalities in production may be associated with inflammation and fibrinolysis at sites of tissue damage such as the atherosclerotic plaque.PAI-2 gene variants have been described: variant A consists of Asn120, Asn404 and Ser413 and variant B consists of Asp120, Lys404 and Cys413. We designed a PCR-RFLP assay using primers spanning the region containing Asn/Lys404 and Ser/Cys413. Variant B contains an Mwol restriction site. We analysed 302 Pima Indians and 286 healthy Caucasian volunteers. To investigate relationships between genotype and vascular disease we analysed 333 Caucasian patients undergoing coronary angiography.Gene variant B was more common in the Pimas than in Caucasians (p <0.0001). There was no significant difference in genotype distribution between the volunteers and patients. In the patients there was no association between genotype and either a history of MI or extent of coronary atheroma.

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Green Synthesis and Spectroscopic Studies of Ag-rGO Nanocomposites for Highly Selective Mercury (II) Sensing

Nanoscience &Nanotechnology-Asia ◽

10.2174/2210681207666170705143629 ◽

2018 ◽

Vol 9 (1) ◽

pp. 101-108 ◽

Cited By ~ 1

Author(s):

Shubhangi J. Mane-Gavade ◽

Sandip R. Sabale ◽

Xiao-Ying Yu ◽

Gurunath H. Nikam ◽

Bhaskar V. Tamhankar

Keyword(s):

Green Synthesis ◽

Color Change ◽

Limit Of Detection ◽

Aqueous Media ◽

Suitable Condition ◽

Cost Effective ◽

Spectroscopic Studies ◽

Calibration Plot ◽

First Time

Introduction: Herein we report the green synthesis and characterization of silverreduced graphene oxide nanocomposites (Ag-rGO) using Acacia nilotica gum for the first time. Experimental: We demonstrate the Hg2+ ions sensing ability of the Ag-rGO nanocomposites form aqueous medium. The developed colorimetric sensor method is simple, fast and selective for the detection of Hg2+ ions in aqueous media in presence of other associated ions. A significant color change was noticed with naked eye upon Hg2+ addition. The color change was not observed for cations including Sr2+, Ni2+, Cd2+, Pb2+, Mg2+, Ca2+, Fe2+, Ba2+ and Mn2+indicating that only Hg2+ shows a strong interaction with Ag-rGO nanocomposites. Under the most suitable condition, the calibration plot (A0-A) against concentration of Hg2+ was linear in the range of 0.1-1.0 ppm with a correlation coefficient (R2) value 0.9998. Results & Conclusion The concentration of Hg2+ was quantitatively determined with the Limit of Detection (LOD) of 0.85 ppm. Also, this method shows excellent selectivity towards Hg2+ over nine other cations tested. Moreover, the method offers a new cost effective, rapid and simple approach for the detection of Hg2+ in water samples.

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Pitfalls in the Immunochemical Determination of β‑Lactam Antibiotics in Water

Antibiotics ◽

10.3390/antibiotics10030298 ◽

2021 ◽

Vol 10 (3) ◽

pp. 298

Author(s):

Alexander Ecke ◽

Rudolf J. Schneider

Keyword(s):

Limit Of Detection ◽

Cost Effective ◽

Enzyme Linked Immunosorbent Assay ◽

Degree Of Hydrolysis ◽

Site Analysis ◽

Antibody Recognition ◽

Immunochemical Determination ◽

Key Factor ◽

Lactam Ring

Contamination of waters with pharmaceuticals is an alarming problem as it may support the evolution of antimicrobial resistance. Therefore, fast and cost-effective analytical methods for potential on-site analysis are desired in order to control the water quality and assure the safety of its use as a source of drinking water. Antibody-based methods, such as the enzyme-linked immunosorbent assay (ELISA), can be helpful in this regard but can also have certain pitfalls in store, depending on the analyte. As shown here for the class of β-lactam antibiotics, hydrolysis of the β‑lactam ring is a key factor in the immunochemical analysis as it influences antibody recognition. With the antibody used in this study, the limit of detection (LOD) in the immunoassay could be significantly reduced by hydrolysis for the five tested penicillins, with the lowest LOD for carbenicillin (0.2 nmol/L) and the greatest impact on penicillins G and V (reduction by 85%). In addition to enhanced quantification, our strategy also provides access to information about the degree of hydrolysis in water samples as shown for the most abundant penicillin amoxicillin.

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