Stabilized, Freeze-Dried PCR Mix for Detection of Mycobacteria

Klatser, P. R.; Kuijper, Sjoukje; Ingen, W. Van; Kolk, A. H. J.

doi:10.1128/jcm.36.6.1798-1800.1998

Cited by 59 publications

(47 citation statements)

References 5 publications

(10 reference statements)

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“…However, because it is a 'hot-start' enzyme, KAPA 2G Fast is best for frozen or lyophilized reactions. If premixed PCR reagents include 5% trehalose, they can be lyophilized and retain activity for up to 1 year at 4°C and for 3 months at 37°C (Klatser et al 1998). We have also found that sequencing reagents dried in 5% trehalose can be stored at 20°C for 4 months without negative impacts on sequence quality.…”

Section: Conclusion: Practical Applications and The Future Of Expresmentioning

confidence: 80%

Express barcodes: racing from specimen to identification

Ivanova

Borisenko

Hebert

2009

Molecular Ecology Resources

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Although devices combining microfluidic and advanced sequencing technologies promise a future where one can generate a DNA barcode in minutes, current analytical regimes typically involve workflows that extend over 2 days. Here we describe simple protocols enabling the advance from a specimen to barcode-based identification in less than 2 h. The protocols use frozen or lyophilized reagents that can be prepackaged into 'kits' and support barcode analysis across the animal kingdom. The analytical procedure allows 5 min for DNA extraction, 25 min for polymerase chain reaction amplification of the barcode region, 25 min for cycle-sequencing, 10 min for cleanup, 45 min for capillary sequencing and 5 min for trace file analysis to complete DNA-based identification. This study involved the comparison of varied DNA preservation and extraction methods, and evaluated Taq polymerases with high processivity and resistance to inhibitors.

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Section: Conclusion: Practical Applications and The Future Of Expresmentioning

confidence: 80%

Express barcodes: racing from specimen to identification

Ivanova

Borisenko

Hebert

2009

Molecular Ecology Resources

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“…and Salmonella spp. in clinical samples (9,10,11,12), and stabilised the PCR reagents for up to one year at -20ºC. Qu et al (13) developed lyophilised thermostable real-time PCR reagents for the detection of Yersinia spp.…”

Section: Discussionmentioning

confidence: 99%

Comparison of stabilisers for development of a lyophilised multiplex reverse-transcription PCR mixture for rapid detection of foot and mouth disease virus serotypes

Sharma¹,

Mahajan²,

Das³

et al. 2014

Rev. Sci. Tech. OIE

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Multiplex reverse-transcription polymerase chain reaction (mRT-PCR) assay is a sensitive and rapid method for the detection and serotyping of foot and mouth disease virus (FMDV). However, the method has not been used to its full potential, because of factors such as cost, a lack of infrastructure and the complexity of the reaction mixture. This study was undertaken to optimise and validate a thermostable, lyophilised, ready-to-use mRT-PCR kit for the rapid detection of FMDV in field laboratories in India. Trehalose, PEG-8000 and glycerol were evaluated for stabilisation of the PCR mixture at ambient temperatures. The lyophilised mRT-PCR kit was validated and found robust enough for use in field-level laboratories. The PCR reaction mixture in the ready-to-use kit has low complexity, so chances of cross-contamination during the preparation of the mixture are limited, but may easily be monitored by using lyophilised internal positive and negative controls. In addition, the requirement to maintain live FMDV isolates as internal positive controls at field-level regional laboratories is eliminated. KeywordsFoot and mouth disease -India -Lyophilised RT-PCR reagent -Ready-to-use RT-PCR kit.

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“…Immobilization of the master mixes increased the stability of the PCR reagents and also made the assays user-friendly, with a requirement to just add the template DNA and make up the reaction volume with sterile water prior to loading on the real-time PCR equipment. Klatser et al (1998) have described a conventional PCR for the detection of mycobacteria using trehalose-based immobilized master mix. However, no study has been carried out to find out whether it is suitable for the real-time PCR.…”

Section: Resultsmentioning

confidence: 99%

Real-time PCR-based rapid and culture-independent detection of Salmonella in dairy milk - addressing some core issues

Syed

Verma

Qazi

2013

Lett Appl Microbiol

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Significance and Impact of the Study: With the familiarity of the polymerase chain reaction (PCR) in diagnostic microbiology, it is becoming increasingly possible to use the nucleic acid amplification methodologies for rapid detection of pathogens under field conditions. However, it is imperative to develop assays that are culture-independent, cost-effective and user-friendly. The methodologies developed in this study, particularly the immobilization of the reaction components on the vessels and total DNA extraction from milk, may help to facilitate the use of this technology under field settings. The protocols may thus facilitate the routine culture-independent detection of pathogens, in particular Salmonella, in dairy milk. AbstractIn this study, methodologies were developed for cost-effective, rapid and userfriendly culture-independent detection of Salmonella in milk by real-time PCR. The SYBR Green-based real-time PCR assay was standardized with primers targeting the Salmonella enterotoxin gene (stn) that have been earlier used for its detection by conventional PCR. Inclusivity tests generated the specific amplifications with a Tm corresponding to 81 AE 0Á5°C. The specificity of the reaction was evaluated with a panel of 36 non-Salmonella strains. Standard curves generated, with different number of cells of this organism in milk, depicted the detection of five cells with a C T value of 37Á17 (SD 0Á43). To make the assays user-friendly and suitable for field applications, protocols were also established for the immobilization of the SYBR Green reaction mixes in the reaction tubes. The immobilized master mixes were stable at 25°C for 4 months and at 8°C for over 6 months. Total DNA was prepared from 150 samples of full-fat dairy milk and subjected to real-time PCR detection wherein 31 samples tested positive for Salmonella. The time of analysis was <5 h.

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Stabilized, Freeze-Dried PCR Mix for Detection of Mycobacteria

Cited by 59 publications

References 5 publications

Express barcodes: racing from specimen to identification

Express barcodes: racing from specimen to identification

Comparison of stabilisers for development of a lyophilised multiplex reverse-transcription PCR mixture for rapid detection of foot and mouth disease virus serotypes

Real-time PCR-based rapid and culture-independent detection of Salmonella in dairy milk - addressing some core issues

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