Comparison of Four Rapid DNA Extraction Techniques for Conventional Polymerase Chain Reaction Testing of Three Mycobacterium spp. that Affect Birds

10.1637/7070 ◽  
2003 ◽  
Vol 47 (4) ◽  
pp. 1486-1490 ◽  
Author(s):  
Lisa A. Tell ◽  
Janet Foley ◽  
Martha L. Needham ◽  
Richard L. Walker
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Conventional Polymerase Chain Reaction ◽  
Extraction Techniques ◽  
Chain Reaction ◽  
Polymerase Chain Reaction Testing ◽  
Polymerase Chain ◽  
Mycobacterium Spp

Detection of Thermophilic Campylobacter spp. in Blood-Free Enriched Samples of Inoculated Foods by the Polymerase Chain Reaction

2000 ◽  
Vol 63 (3) ◽  
pp. 299-303 ◽  
Author(s):  
RICHARD L. THUNBERG ◽  
TONY T. TRAN ◽  
MARK O. WALDERHAUG
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Raw Milk ◽  
Extraction Techniques ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Enrichment Broth ◽  
Thermophilic Campylobacter ◽  
Polymerase Chain ◽  
Campylobacter Spp

The detection of thermophilic Campylobacter spp., as represented by Campylobacter jejuni, by the polymerase chain reaction (PCR) was investigated and compared with the selective agar isolation (SAI) method. Stationary-phase cultures of C. jejuni were inoculated into either blood-free enrichment broth (BFEB) or BFEB that contained 10% broccoli, crabmeat, mushroom, raw milk, and raw oyster rinses. Following a 48-h enrichment period, aliquots of food test portions were removed for simultaneous analysis by PCR and SAI. It was determined that the presence of charcoal and iron in the enrichment broth interfered with the PCR assay. Therefore, three DNA extraction techniques were developed and evaluated using a 16S rRNA primer pair in the PCR assay. The 50% end point (DL50) values (determined upon six initial C. jejuni spiking levels) were used to assess the frequency of isolation utilizing PCR versus SAI for the detection of this organism in the enrichment matrices. There were virtually no differences in detection of C. jejuni among enriched samples analyzed by PCR and SAI. Mean DL50 values (n = 3) for plain BFEB, broccoli, crabmeat, mushroom, raw milk, and raw oyster were, respectively, 0.02 (PCR) versus 0.01 (SAI), 0.01 versus 0.06, 0.07 versus 0.04, 0.03 versus 0.08, 0.01 versus 0.01, and 0.01 versus 0.01 CFU/5 g food. Significant variability in the detection limit of C. jejuni by PCR in the food enrichments was observed among DNA extraction techniques. Using 48-h enrichment cultures followed by PCR analysis could save 1 day of the time required for the presumptive identification of C. jejuni in suspected foods.

Persistent Leptospiruria in Five Dogs Despite Antimicrobial Treatment (2000–2017)

2019 ◽  
Vol 55 (1) ◽  
pp. 42-47 ◽  
Author(s):  
Tara Mauro ◽  
Kenneth Harkin
Keyword(s):  
At Risk ◽  
Polymerase Chain Reaction ◽  
Antimicrobial Treatment ◽  
Renal Tubules ◽  
Chain Reaction ◽  
Polymerase Chain Reaction Testing ◽  
Pet Owners ◽  
Zoonotic Risk ◽  
The Face ◽  
Polymerase Chain

ABSTRACT In dogs with leptospirosis, doxycycline therapy is recommended as the preferred therapy for its ability to eliminate the organism from all tissues, including the renal tubules. Elimination of organisms from the renal tubules terminates leptospiruria and prevents transmission of the organism. This report describes the discovery of persistent leptospiruria in the face of therapy with doxycycline in four dogs and enrofloxacin in one dog. Leptospiruria was confirmed by polymerase chain reaction testing for pathogenic leptospires in all five dogs. In two dogs, leptospiruria resolved after a change in therapy to enrofloxacin. In three dogs, doxycycline and/or enrofloxacin were ineffective at eliminating leptospiruria, which then resolved after therapy with clarithromycin. Pet owners could be at risk as persistent leptospiruria poses a potential zoonotic risk. The potential reasons for persistent leptospiruria as demonstrated by polymerase chain reaction testing are discussed.

scholarly journals UTILITY OF ANTIGEN DETECTION TEST AND POLYMERASE CHAIN REACTION IN THE DIFFERENTIATION OF TUBERCULOUS AND NON-TUBERCULOUS MYCOBACTERIA

2017 ◽  
Vol 10 (6) ◽  
pp. 289
Author(s):  
Yogita Singh ◽  
Raji Vasanth ◽  
Shrikala Baliga ◽  
Dhanashree B
Keyword(s):  
Polymerase Chain Reaction ◽  
False Negative ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Chain Reaction ◽  
College Hospital ◽  
Medical College ◽  
Negative Results ◽  
Polymerase Chain ◽  
Mycobacterium Spp

Objectives: Cultivation and identification of mycobacteria to species level remains difficult and time-consuming. Hence, easy and rapid diagnostic methods are necessary for the differentiation of Mycobacterium tuberculosis (MTB) from non-tuberculous mycobacteria (NTM). The present study aims to detect and differentiate MTB from NTM isolated from clinical samples by immunochromatographic test (ICT) and polymerase chain reaction (PCR). Methods: Over a period of 1 year, clinical samples (n=496) received from suspected cases of TB, at the Department of Microbiology, Kasturba Medical College Hospital, Mangalore were cultured to isolate Mycobacterium spp. Identification of all the isolates was done by conventional biochemical technique, ICT, and PCR. Results: Among the 496 samples processed, 49 (9.87%) were acid-fast bacilli smear positive and 59 (11.89%) samples showed the growth of Mycobacterium spp. Among these, 10 were rapid growers, 49 were slow-growing mycobacteria, out of which 30 were MTB as identified by conventional biochemical reaction. Out of 59 Mycobacterial isolates subjected to ICT for the detection of MPT 64 antigen, only 28 were identified as MTB. However, all the 30 isolates were correctly identified as MTB by PCR. Conclusion: Hence, PCR is essential for rapid differentiation of non-tuberculous Mycobacterium from MTB. False negative results seen with immunochromatographic MPT 64 antigen assay could be due to mutations within the mpt64 gene. Further studies are necessary to characterize these PCR-positive and immunochromatographic assay negative MTB isolates.

scholarly journals Impact of Multiplex Polymerase Chain Reaction Testing for Respiratory Pathogen Detection in Pediatric Patients

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S503-S503
Author(s):  
Courtney C Sutton ◽  
Patti J Walton ◽  
Montgomery F Williams ◽  
Tracey L Bastian ◽  
Michael Wright ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Pathogen Detection ◽  
Pediatric Patients ◽  
Respiratory Pathogen ◽  
Chain Reaction ◽  
Polymerase Chain Reaction Testing ◽  
Polymerase Chain

scholarly journals Detection of MYCN Amplification in Serum DNA Using Conventional Polymerase Chain Reaction

2016 ◽  
Vol 31 (9) ◽  
pp. 1392 ◽  
Author(s):  
Youngeun Ma ◽  
Ji Won Lee ◽  
Soo Jin Park ◽  
Eun Sang Yi ◽  
Young Bae Choi ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Conventional Polymerase Chain Reaction ◽  
Mycn Amplification ◽  
Chain Reaction ◽  
Serum Dna ◽  
Polymerase Chain

scholarly journals Method Comparison of DNA Isolation and Quantification for Fish and Seafood Authenticity Determination

2018 ◽  
Vol 13 (3) ◽  
pp. 115
Author(s):  
Dwiyitno Dwiyitno ◽  
Stefan Hoffman ◽  
Koen Parmentier ◽  
Chris Van Keer
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Blue Mussel ◽  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Seafood Products ◽  
Polymerase Chain ◽  
Commercial Kit

Fish and seafood products has been commonly targeted for fraudulent activities. For that reason, authentication of fish and seafood products is important to protect consumers from fraudulent and adulteration practices, as well as to implement traceability regulation. From the viewpoint of food safety, authenticity is beneficial to protect public from serious food poisoning incidents, such as due to ingestion of toxic species. Since DNA based identification depends on the nucleic acid polymerase chain reaction (PCR), the quantity and quality/purity of DNA will contribute significantly to the species authentication. In the present study, different DNA extraction and purification methods (3 classical methods and one commercial kit) were compared to produce the better isolated DNA for PCR amplification. Additionally, different methods for the estimation of DNA concentration and purity which is essential for PCR amplification efficiency were also evaluated. The result showed that classical DNA extraction methods (based on TNES-Urea) yielded a higher amount of DNA (11.30-323.60 ng/g tissue) in comparison to commercial kit/Wizard Promega (5.70-83.45 ng/g tissue). Based on the purity of DNA extract (A260/280), classical DNA extraction method produced relatively similar on DNA quality to the commercial kit (1.79-2.12). Interestingly, all classical methods produced DNA with A260/280 ratio of more than 2.00 on the blue mussel, in contrast with commercial kit. The commercial kit also produced better quality of DNA compared to the classical methods, showing the higher efficiency in PCR amplification. NanoDrop is promising as cheap, robust and safe UV-spectrophotometer method for DNA quantification, as well as the purity evaluation.Keywords: seafood authenticity, DNA isolation, polymerase chain reaction, NanoDrop, Picogreen

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