The MaxSignal® Listeria Extraction and Detection Kit uses an end-point PCR assay to screen for the presence of Listeria bacteria in potentially infectious material. The purification method is based on lysis of Listeria pellets at high temperature followed by solid-phase extraction of the DNA on silica filters. The procedure can be carried out in less than 30 min using common laboratory equipment. The use of purified Listeria DNA allows for increased sensitivity compared to Listeria detection methods that use samples consisting of unpurified bacterial lysates, because the DNA is concentrated into a smaller volume for use in volume-limited PCR reactions. The kit also contains 3 synthetic templates for use as Internal Amplification Controls. The synthetic templates have primer binding sites for one of the Outer Primer Pairs (A, B, or C) and an internal region consisting of a plasmid DNA sequence. The plasmid sequence is the same in all 3 templates. The control templates are typically added to the PCRs along with the sample DNA, to create positive amplification controls. Amplification of the expected size amplicons from the synthetic templates proves that the sample DNA did not contain inhibitors of PCR. To maximize user convenience, the reagents for PCR (buffer, dNTPs, and thermostable polymerase) are provided as a 5x MasterMix. The MasterMix also contains components to allow the PCR to be directly loaded for gel analysis, without adding gel-loading solution. The kit includes a molecular weight “ladder” for analysis of results from experimental samples. The kit contains 6 pairs of oligonucleotide primers targeting outer and nested regions of three different types of Listeria sample: 2 primer pairs specific for all Listeria species (Primer Pairs A outer and A nested); 2 primer pairs specific for Listeria monocytogenes (Primer Pairs B outer and B nested); and 2 primer pairs specific for Listerial moncytogenes isolate 4b (Primer Pairs C outer and C nested). The primers were designed against Listeria target genes. The kit also includes reagents and materials for purification of Listeria DNA from liquid cultures.
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