scholarly journals Comparison of single-round polymerase chain reaction (PCR) and pepsin-trypsin digest (PTD) methods for detection of Myxobolus cerebralis

2001 ◽  
Vol 45 ◽  
pp. 109-114 ◽  
Author(s):  
GJ Schisler ◽  
EP Bergersen ◽  
PG Walker ◽  
J Wood ◽  
JK Epp
Keyword(s):  
Polymerase Chain Reaction ◽  
Myxobolus Cerebralis ◽  
Chain Reaction ◽  
Round Polymerase Chain Reaction ◽  
Polymerase Chain

Quantum dots for a high-throughput Pfu polymerase based multi-round polymerase chain reaction (PCR)

The Analyst ◽  
2018 ◽  
Vol 143 (5) ◽  
pp. 1259-1267 ◽  
Author(s):  
Fuming Sang ◽  
Zhizhou Zhang ◽  
Lin Yuan ◽  
Deli Liu
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantum Dots ◽  
High Throughput ◽  
Chain Reaction ◽  
Round Polymerase Chain Reaction ◽  
Polymerase Chain

We developed a Pfu polymerase based multi-round PCR technique assisted by quantum dots (QDs).

Nanogold-Assisted Multi-Round Polymerase Chain Reaction (PCR)

2007 ◽  
Vol 7 (12) ◽  
pp. 4428-4433 ◽  
Author(s):  
Jiakui Pan ◽  
Haikuo Li ◽  
Xueyan Cao ◽  
Jiehuan Huang ◽  
Xiaodong Zhang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Gel Electrophoresis ◽  
Genomic Dna ◽  
Target Product ◽  
Chain Reaction ◽  
Round Polymerase Chain Reaction ◽  
Bright Band ◽  
Target Dna ◽  
Specific Amplification ◽  
Polymerase Chain

We have previously demonstrated that nanogold effectively enhances the specificity and yield of error-prone two-round polymerase chain reaction (PCR). Here we reported that, with the assistance of nanogold, we could perform multi-round PCR. In the presence of appropriate amount of 10 nm nanogold, we could obtain the target product even after six rounds of PCR, as manifested by a single bright band in gel electrophoresis (1% agarose). In fact, we could still observe the target band even at the 7th round of PCR, which nevertheless was accompanied by smearing bands (non-specific amplification). In contrast, in the absence of nanogold, the target band was completely lost only after four rounds of amplification. This marked difference in the performance of multi-round PCR clearly showed that nanogold was a powerful enhancer for PCR. More importantly, with this nanogold-assisted multi-round PCR, it might be possible to produce a large amount of target DNA, or to amply very low copies of genomic DNA from rare sources.

scholarly journals Evaluation of Nested Polymerase Chain Reaction, Pepsin/Trypsin Digest, and Histology for the Detection of Myxobolus Cerebralis in Salmonid Fishes of Indiana and Michigan

2002 ◽  
Vol 14 (3) ◽  
pp. 251-254 ◽  
Author(s):  
T. Qureshi ◽  
M. R. White ◽  
C. Santrich
Keyword(s):  
Polymerase Chain Reaction ◽  
Nested Pcr ◽  
True Positive ◽  
Myxobolus Cerebralis ◽  
Chain Reaction ◽  
False Negatives ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Low Prevalence ◽  
Pcr Test

The objectives of this study were to survey fish from state hatcheries in Indiana and Michigan and to compare the nested polymerase chain reaction (PCR) test with pepsin/trypsin digest (PTD) and histopathology for the diagnosis of whirling disease (WD). One group of 40 and 9 groups of 60 fish heads, for a total of 580 samples, were submitted from hatcheries in Indiana and Michigan. These samples were examined for myxozoan spores using histopathology, PTD, and PCR tests. The heads were hemisectioned, and one half was fixed in 10% neutral-buffered formalin for histopathologic examination. The other half was processed for PTD. Some of the sediment was examined for the presence of myxozoan spores, and the rest was prepared for the nested PCR. Histologic examinations did not reveal Myxobolus cerebralis in any of the 580 samples. One hundred serial step sections, taken at 5-μm intervals, were evaluated for samples with positive spore identification by PTD. Histologic examination of these sections failed to reveal any myxozoan parasites. Myxozoan spores were observed in 16.9% (98/580) of samples in sediment after PTD. Spores morphologically similar to those of M. cerebralis were observed in 1.0% of PTD samples ( n = 6). The nested PCR indicated that M. cerebralis spores were present in 0.5% of samples ( n = 3). All 3 nested PCR-positive samples came from the same hatchery; however, spores of M. cerebralis were seen in 1 sample, spores of other myxozoan species were seen in the second sample, and spores were not seen in the third sample. When comparing the PTD to the nested PCR test, the PTD diagnosed 1 true positive, 5 false positives, 2 false negatives, and 572 true negatives, for a sensitivity of 33% and a specificity of 99.1%. Screening for M. cerebralis infection in this study indicated a low prevalence of the disease. Histopathology was a very insensitive indicator of WD. The PCR test was highly specific and was used to differentiate spores of M. cerebralis from similar spores of other species.

Diagnosis of Trichophyton rubrum from Onychomycotic Nail Samples Using Polymerase Chain Reaction and Calcofluor White Microscopy

2008 ◽  
Vol 98 (3) ◽  
pp. 224-228 ◽  
Author(s):  
Aditya K. Gupta ◽  
Muhammad Zaman ◽  
Jagpal Singh
Keyword(s):  
Polymerase Chain Reaction ◽  
Fluorescence Microscopy ◽  
Polymerase Chain Reaction Assay ◽  
Potassium Hydroxide ◽  
Trichophyton Rubrum ◽  
Actin Gene ◽  
Chain Reaction ◽  
Calcofluor White ◽  
Round Polymerase Chain Reaction ◽  
Polymerase Chain

Background: A high rate of false-negative dermatophyte detection is observed when the most common laboratory methods are used. These methods include microscopic observation of potassium hydroxide–digested nail clippings and culture methods using agar-based media supplemented with cycloheximide, chloramphenicol, and gentamicin to isolate dermatophytes. Microscopic detection methods that use calcofluor white staining or periodic acid–Schiff staining may also be substituted for and have previously been reported to be more sensitive than potassium hydroxide–digested nail clippings. Methods: Trichophyton rubrum infections were detected directly from nails in a double-round polymerase chain reaction assay that uses actin gene–based primers. This method was compared with detection of fungal hyphae by using calcofluor white fluorescence microscopy of nail samples collected from 83 patients with onychomycosis who were undergoing antifungal drug therapy. Results: Twenty-six of 83 samples (31.3%) were found to be positive by calcofluor white fluorescence microscopy, and 21 of 83 samples (25.3%) yielded positive results for T rubrum when actin gene–based primers in a double-round polymerase chain reaction assay were used. When calcofluor white fluorescence microscopy and polymerase chain reaction assay were used, the combined detection was 46.9% compared with 31.3% when calcofluor microscopy and culture of nail samples on Sabouraud’s dextrose agar supplemented with cycloheximide, chloramphenicol, and gentamicin were used. Conclusions: These results suggest that the use of a direct DNA protocol is an alternative method for detecting Trichophyton infections. When this protocol is used, the presence of T rubrum DNA is directly detected. However, the viability of the dermatophyte is not addressed, and further methods need to be developed for the detection of viable T rubrum directly from nail samples. (J Am Podiatr Med Assoc 98(3): 224–228, 2008)

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