Long-Term Storage of Unamplified Complete PCR Mixtures

Reischl, Udo; Haber, B.; Vandezande, Wim; Baère, Thierry De; Vaneechoutte, Mario

doi:10.2144/97234bm03

Cited by 7 publications

(5 citation statements)

References 7 publications

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“…d Each set of test samples included a negative control tube in which water was substituted for a test sample. A complete mastermix was prepared for each primer set, and 40-l aliquots were introduced into the reaction tubes, g which were held at Ϫ20 C until use (after this study was concluded, the holding temperature was changed to Ϫ70 C, as previously recommended 18 ). Positive control DNA (10 ng) was obtained from bacterial culture extracts.…”

Section: Methodsmentioning

confidence: 99%

Polymerase Chain Reaction Identification of Mycobacterium Avium in Formalin-Fixed, Paraffin-Embedded Animal Tissues

1999

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Abstract.A PCR procedure previously developed for identification of Mycobacterium bovis in formalinfixed tissues was used to identify mycobacteria of the M. avium complex. Tissues were examined from 100 culture-positive cases of M. avium complex infection, including 86 in which the subspecies was not identified and 14 that had been identified as M. avium subsp. paratuberculosis. Each sample was tested with 5 primer sets, 16S ribosomal RNA (rRNA), IS900, IS901, IS1245, and a heat shock protein (hspX), that detect 1 or both M. avium subspecies. The success rate of PCR detection varied with the primers used and the animal species tested. Among the 86 cases with no M. avium subspecies designation, primers for the 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted with any other primer set. The overall detection rate in this group of samples was 71%: highest in avian tissues (89%) followed by swine (72%) and ruminants (57%) None of the avian or swine tissues reacted with primers for IS900 or hspX, which identify M. a. paratuberculosis. In contrast, 7 of the 12 ruminant samples that were 16S rRNA positive reacted with 1 or both of these primers. All of the 14 cases shown by culture to be M. a. paratuberculosis infections were positive with IS900 primers, whereas only 11 were positive for 16S rRNA. These results indicate that 16S rRNA primers are the most useful for PCR identification of M. avium in formalin-fixed tissues of nonruminant species. However, IS900 primers should also be used when ruminant tissues are examined because these primers provide the greatest sensitivity for detection of M. a. paratuberculosis infections. Eradication of bovine tuberculosis in the UnitedStates has been a long-term goal that is now nearing attainment. Early in the campaign, the major effort was directed toward identification of infected herds by systematic skin testing. As incidence of the disease declined, the focus of attention shifted to a surveillance program that is based on detection of suspect lesions in slaughter animals, with subsequent histopathologic examination and culture of the tissues. If culture results indicate the presence of Mycobacterium bovis, epidemiologic investigations are begun to locate the potential source of infection and to identify other animals that might have been exposed. Unfortunately, several weeks may elapse before this effort can be initiated because M. bovis is such a slow-growing organism. To provide a more rapid diagnostic method, a polymerase chain reaction (PCR) procedure was developed that allows specific identification of M. tuberculosis complex bacteria (M. tuberculosis, M. bovis, M. africanum, and M. microti) in formalin-fixed, paraffin-embedded tissues. 14 When positive results are obtained with this procedure, it is possible to make a

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Section: Methodsmentioning

confidence: 99%

Polymerase Chain Reaction Identification of Mycobacterium Avium in Formalin-Fixed, Paraffin-Embedded Animal Tissues

1999

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“…14 RAPD/AP primers: HLWL74 (5 0 -ACGTATCTGC-3 0 ) (Grattard et al, 1994) (Sakallah et al, 1995), primer AP (=''primer 2'') (5 0 -GGTGGTGGCT-3 0 ) (Grattard et al, 1996;Reischl et al, 1997), primer C (5 0 -AGTCAGCCAC-3 0 ) and primer D (5 0 -AATCGGGCTG-3 0 ) (Bansal and McDonell, 1997), primer 10 (=primer L) 5 0 -TGCGGACCTG-3 0 ) (Sandery et al, 1994) Lus et al, 1993) and BG2 (5 0 -TACATTCGAG-GACCCCTAAGTG-3 0 ) (Van Belkum et al, 1993). 2 ERIC primers: ERIC2 (5 0 -AAGTAAGT-GACTGGGGTGAGCG-3 0 ) and ERIC IR-1 (5 0 -ATG-TAAGCTCCTGGGGATTCAC-3 0 ) (Van Belkum et al, 1996).…”

Section: Dna Fingerprinting Using Pcrmentioning

confidence: 99%

Evaluation of different primers for DNA fingerprinting of Legionella pneumophila serogroup 1 strains by polymerase chain reaction

Wiese

Helbig

Lück

et al. 2004

International Journal of Medical Microbiology

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“…Exposure to elevated temperatures, which is commonly employed for ameliorating issues of inhibitory secondary structure as well as during thermal cycling, further promotes this decomposition. This is also an issue with long-term storage of dNTPs, leading to a commonly observed failure of PCR when older stocks of dNTPs are used (11). Furthermore, the byproduct of DNA synthesis, pyrophosphate, can also act to inhibit primer extension when its concentration builds over time.…”

Section: Introductionmentioning

confidence: 99%

Polymerase synthesis of four-base DNA from two stable dimeric nucleotides

Mohsen

Kool

2019

Nucleic Acids Research

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We document the preparation and properties of dimerized pentaphosphate-bridged deoxynucleotides (dicaptides) that contain reactive components of two different nucleotides simultaneously. Importantly, dicaptides are found to be considerably more stable to hydrolysis than standard dNTPs. Steady-state kinetics studies show that the dimers exhibit reasonably good efficiency with the Klenow fragment of DNA polymerase I, and we identify thermostable enzymes that process them efficiently at high temperature. Experiments show that the dAp5dT dimer successfully acts as a combination of dATP and dTTP in primer extension reactions, and the dGp5dC dimer as a combination of dGTP and dCTP. The two dimers in combination promote successful 4-base primer extension. The final byproduct of the reaction, triphosphate, is shown to be less inhibitory to primer extension than pyrophosphate, the canonical byproduct. Finally, we document PCR amplification of DNA with two dimeric nucleotides, and show that the dimers can promote amplification under extended conditions when PCR with normal dNTPs fails. These dimeric nucleotides represent a novel and simple approach for increasing stability of nucleotides and avoiding inhibition from pyrophosphate.

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Long-Term Storage of Unamplified Complete PCR Mixtures

Cited by 7 publications

References 7 publications

Polymerase Chain Reaction Identification of Mycobacterium Avium in Formalin-Fixed, Paraffin-Embedded Animal Tissues

Polymerase Chain Reaction Identification of Mycobacterium Avium in Formalin-Fixed, Paraffin-Embedded Animal Tissues

Evaluation of different primers for DNA fingerprinting of Legionella pneumophila serogroup 1 strains by polymerase chain reaction

Polymerase synthesis of four-base DNA from two stable dimeric nucleotides

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