Citation:

Vesper, S J. AND R. A. Haughland. 21ST CENTURY MOLD ANALYSIS IN FOOD. FOOD QUALITY, 39-40, (2003).

Impact/Purpose:

To understand children's risks from exposure to molds in their environment and to explore risk management options for mitigating those risks.

Description:

Traditionally, the indoor air community has relied on mold analysis performed by either microscopic observations or the culturing of molds on various media to assess indoor air quality. These techniques were developed in the 19th century and are very laborious and time consuming with distinct limitations in standardization. Today, the analytical laboratory has a 21st century solution to mold identification and enumeration.
In 1993, the Nobel Prize was awarded for the development of a method to amplify DNA sequences using the enzyme DNA polymerase. DNA sequences are those strings of A, C, G and Ts that form the genomic blueprint that make each creature, including each mold species, unique. This process of DNA amplification became known as the polymerase chain reaction or PCR.
The PCR process is initiated by short, specific sequences of DNA called the forward and reverse primers. As PCR amplification proceeds, copies of DNA accumulate and the number of accumulated copies can be estimated. PCR technology, as practiced before 1997, had great specificity but it lacked good quantitation and was relatively slow to perform. In 1997, Applied Biosystems, Inc. in Foster City CA announced the release of a new instrument called the Sequence Detector which provided a very accurate way to measure the accumulated PCR product. This measurement was accomplished by adding a species specific, fluorescently labeled probe in the sequence between the primers used in PCR. This process now goes by the name quantitative PCR (QPCR).
The QPCR analysis begins by extracting the DNA from an environmental sample. For example, a dust sample is weighed and added to a 2 ml extraction tube that contains tiny glass beads and an extraction fluid. The extraction tube is then shaken for 1 minute at 5,000 rpms which releases the DNA from the cells. For an air sample collected on a filter, the entire filter is added to the extraction tube and shaken. The extracted DNA is then ready for analysis or it made need some additional purification depending on how "dirty" it is. With a few simple steps, the purified sample is ready for analysis. Next the analyst needs the mold specific primers and probe.
In May of 2002, EPA was granted a patent (6,387,652) on the DNA sequences of primers and probe needed for analyzing well over 100 of the most important or numerous indoor molds. The EPA has now licensed 10 companies (Table 1) in the US and UK to use these primers and probes in QPCR analysis. Now an analyst from one of these licensed analytical laboratories can go to the freezer, where the primers and probes are stored, and select the set for each of the target mold species.
Of course, by this time, the analytical laboratory has already invested $40 to $100 K to purchase a Sequence Detector instrument. (There are now a number of manufactures of these instruments and price depends on many factors including the number of analyses that can be run at the same time). By this time, the analytical laboratory has also prepared standard spore suspensions, containing known numbers of spores, of each of the molds.
The analyst adds the primers and probe for a particular species together with the amplification solution that contains the PCR polymerase to form the reaction mixture. To this mixture is added a small aliquot of the purified sample DNA extract. This process is repeated for each of the target species. At the same time, the analyst prepares an extract of the standard spore suspension for the particular target mold(s).
The reaction mixtures are then placed in the sequence detector instrument which causes the mixture to heat which opens the double helix of the sample DNA and allows the primers and probe to hybridize or bind, if a match is found in the sample. If there is an exact match, fluorescence from the probe is measured. The sequence detector records and sums this fluorescence from each reaction mixture. At the same time, this analysis is performed with the standard spore suspension with known number of spores of the target mold. By comparing the fluorescence from the standard spore suspension analysis to the fluorescence of the DNA extracted from the environmental sample, the analyst can determine the number of target spores that was in the environmental sample. This process is repeated for as many molds as desired.
QPCR-based mold analysis has many advantages. QPCR provides simple, standardized speciation of molds. Air sampling can be extended for hours to days, if desired. And QPCR results can be obtained in a matter of hours not weeks. At this time, QPCR is more expensive than other methods of mold analysis but, like an new technology, costs will decline with greater volume of use. If we are ever going to understand the role of molds in human health, we need the standardized method of analysis which QPCR provides.

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