Method of identifying and quantifying specific fungi and bacteria

[0002] The present application is a continuation in part of Ser. No. 09/290,990, filed Apr. 14, 1999 now abanonded, which claims priority from provisional application Ser. No. 60/081,773, filed Apr. 15, 1998, the entire contents of both of which are hereby incorporated by reference.

[0003] The present invention relates to a method of identifying and quantifying specific fungi and bacteria using specific DNA sequences, as described and taught herein. These sequences can be used with real time detection of PCR products with a fluorogenic probe system or other molecular approaches like hybridizations.

[0004] Fungi and bacteria are the source of or contribute to many health problems including infections, gastroenteritis, ulcers, asthma, allergies and sinusitis. The rapid identification of these microorganisms is critical for diagnosis and treatment. In addition, detecting and/or quantifying these microorganism in the environment may help to prevent adverse health effects.

[0005] Limitations of Current Technology

[0006] In order to determine the risk fungi and bacteria pose to human health, it is necessary to know what fungi and bacteria are present and in what numbers. Fungi and bacteria can be ingested, inhaled, or might enter the body through abrasions or punctures. It is important to identify these microorganisms as specifically and as rapidly as possible. Some species of a particular genus are harmless whereas others of the same genus may cause significant health effects. So without knowing precisely what microorganisms are present and in what numbers, it is impossible to evaluate the potential for negative health effects or the establishment of a risk assessment.

[0007] In the past, the detection and quantitative measurement of fungi and bacteria in samples has been performed either by direct microscopic examination of the collected cells or by growing cells on a suitable medium and identification and enumeration of the resultant colonies. The first method is highly labor intensive and is subject to potential errors in the recognition and positive identification. The second method is both time consuming and subject to significant quantitative inaccuracy. Both methods require extensive experience on the part of the analyst.

[0008] Some molecular approaches, such as the conventional polymerase chain reaction (PCR) procedure, are subject to inaccuracies due to the difficulty of quantifying the product. This procedure is also relatively slow and requires expertise in molecular biology.

[0009] It is an object of the present invention to overcome the aforesaid deficiencies in the prior art.

[0010] It is an object of the present invention to provide a simple, reliable method for detecting and quantifying some fungi and bacteria by using the nucleotide sequences specific to each species or group of species of fungi and bacteria, as described herein.

[0011] According to the present invention, fungi and bacteria can be identified and quantified by using a nucleotide sequence specific to the particular species or, in the case of some fungi, group of species. Many methods including using real time, probe-based detection of polymerase chain reaction (PCR) products (e.g. TaqMan™ system) or other methods of detection and quantification including hybridization or conventional PCR could be used with these sequences.

[0012] Theory

[0013] Each microorganism is unique because of the sequence of some of the nucleotides in its DNA. However, there are many sequences which are common to more than one organism. There is thus a hierarchy or classification into which all microorganisms can be arranged. The “species” are typically the finest level of distinction that is recognized for separation of different members of a given genus. In the past, species were separated on the basis of morphological or biochemical differences. In order to identify or separate different species on the basis of its DNA sequence, one finds sequences that are unique to a given species but at the same time common to all isolates of a given species.

[0014] For this invention the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA) of the different fungi were used. For the bacteria, the sequences of unique enzymes were chosen.

[0015] To apply this invention, a number of possible detection methods are possible. For example, the TaqMan™, 3′-5′ exonuclease assay signals the formation of PCR amplicons by a process involving the nucleolytic degradation of a double-labeled fluorogenic probe that hybridizes to the target template at a site between the two primer recognition sequences (cf. U.S. Pat. No. 5,876,930). The model 7700 automates the detection and quantitative measurement of these signals, which are stoichiometrically related to the quantities of amplicons produced, during each cycle of amplification In addition to providing substantial reductions in the time and labor requirements for PCR analyses, this technology permits simplified and potentially highly accurate quantification of target sequences in the reactions.

[0016] There are additional systems and other molecular approaches that operate upon essentially the same principal. What is common to all of these technologies is the need for the identification of specific sequences that are unique to the targeted organism but common to all members of the species. The present invention teaches these identifying sequences and gives a description of the practical application of the sequences in the identification and quantification of specific fungi and bacteria.

[0017] FIG. 1 illustrates the sensitivity of the assay of the present invention.

[0018] FIG. 2 shows the actual vs. the expected amounts of conidia detected.

[0019] FIG. 3 shows TaqMan Threshold Responses from ten-fold dilutions of a single DNA extract.

[0020] FIG. 4 shows H. pyloricounts per assay plotted against cycle threshold values.

[0021] DNA Extraction

[0022] Genomic DNA is extracted using standard methods, e.g., the glass bead milling and glass milk adsorption method or any similar procedure of extracting genomic DNA.

[0023] Reactions are prepared in 0.5 ml thin-walled, optical grade PCR tubes (PE Applied Biosystems, Foster City Calif.) by addition of the following components; 12.5 μl of TaqMan Universal Master Mix (a 2×-concentrated, proprietary mixture of AmpliTaq Gold™ DNA polymerase, AmpErase® UNG, dNTPs with UTP, passive reference dye and optimized buffer components, PE Applied Biosystems, Foster City Calif.); 2.5 μl of a mixture of forward and reverse primers (10 nM each); 2.5 μl of 400 nM TaqMan probe; 2.5 μl of 2 mg/ml bovine serum albumin, fraction V (Sigma Chemical, St. Louis, Mo.) and 5 μl of DNA template.

[0024] For each targeted fungus or bacterium, the appropriate forward primer, reverse primer and probe (Tables 1 and 2) are to be obtained. The probe is labeled with an appropriate set of dyes or other markers for the particular system of measurement being used.

[0025] For each target species or group of species, a calibrator sample with a known number of conidia or cells is used as a standard. To ensure that the sample matrix does not affect the PCR reaction and, thus the quantitative results, an internal standard is used. Addition of these conidia or cells to both the test and calibrator samples normalize the target species or group for potential sample to sample variability in DNA extraction efficiencies.

[0026] One skilled in the art will appreciate that primers and/or probes can be used which are not identical to the ones described above, as long as there is substantial similarity between the sequences. For purposes of the present invention, “substantial similarity” means that more than 90-110% of the sequence is the same as the sequences enumerated above.

[0027] Performance of Assay

[0028] Standard procedures for the operation of the model 7700 or similar detection system are used. This includes, for example with the model 7700, use of all default program settings with the exception of reaction volume which was changed from 50 to 25 μl. Thermal cycling conditions consisting of two min at 50° C. 10 min at 95° C., followed by 40 cycles of 15 sec at 95° C. and 1 min at 60° C. Cycle threshold (C_T) determinations, i.e. non-integer calculations of the number of cycles required for reporter dye fluorescence resulting from the synthesis of PCR products to become significantly higher than background fluorescence levels were automatically performed by the instrument for each reaction using default parameters. Assays for fungal target sequences and G. candidum(reference) sequences in the same DNA samples are performed in separate reaction tubes.

[0029] Quantification of Fungal or Bacterial Target

[0030] Quantification is performed by first subtracting mean reference sequence C_Tvalues from mean target sequence C_Tvalues for both test samples and a pre-specified calibrator sample to obtain ΔC_Tvalues. Calibrator sample ΔC_Tvalues are then subtracted from ΔC_Tvalues of the test samples to obtain ΔΔC_Tvalues. Assuming an amplification efficiency of one (i.e. a doubling of the target sequence for each cycle), the ratio of target sequences in the test and calibrator samples is given by 2^−ΔΔCT. (If the efficiency is less than one, then the new amplification efficiency value is used instead of 2.) For example, a ratio of 0.1, calculated in this manner, would indicate that the target sequence level in the test sample is one-tenth the level found in the calibrator sample. A direct comparison (ΔC_T) approach should allow the discrimination of 1-fold differences in the quantities of target sequences in different samples with 95% confidence.

[0031] Quantitative Measurement of Stachybotrys chartarumconidia using Real Time Detection of PCR Products with the TaqMan™ Fluorogenic Probe System

[0032] Conidial stocks of the target fungus, e. g. Stachybotrys chartarum,and the reference target, e.g. Geotrichum candidum,were prepared to act as calibrator and internal standard, respectively.

[0033] Genomic DNAs were extracted from 20 μl conidial suspensions using a glass bead milling and glass milk adsorption method. Briefly, this method involved mixing test and reference conidia suspensions (10 μl ea.) with 0.3 g of acid-washed glass beads (G-1277; Sigma, St. Louis, Mo.) and 10 μl, 100 μl and 300 μl, respectively, of glass milk suspension, lysis buffer and binding buffer from an Elu-Quik DNA purification kit (Schleicher and Schuell, Keene, N.H.) in sterile 2 ml conical bottom, screw cap tubes (506-636; PGC Scientifics, Gaithersburg, Md.). The tubes were shaken in a mini beadbeater (Biospec Products, Bartlesville, Okla.) for one minute at maximum rate and DNAs were recovered in final volumes of 200 μl distilled water after performing a slight modification of the small-scale protocol provided with the Elu-Quik purification kit.

[0034] The TaqMan probes and primers were obtained from the custom oligonucleotide synthesis facility at PE-Applied Biosystems (Foster City, Calif.). TaqMan probes contained a TAMRA group conjugated to their 3′-terminal nucleotide and a FAM group linked to their 5′-terminal nucleotides as the quencher and reporter fluorochromes, respectively. For Geotrichum candidum,the forward primer is NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:86), the reverse primer is GcandR1: 5′-AGAAAAGTTGCCCTCTCCAGTT (SEQ ID NO:87), and the probe is GeoP2: 5′-TCAATCCGGAAGCCTCACTAAGCCATT (SEQ ID NO:88). For Stachybotrys chartarum,the forward primer is StacF4 5′-TCCCAAACCCTTATGTGAACC (SEQ ID NO:186), the reverse primer is StacR5 5′-GTTTGCCACTCAGAGAATACTGAAA (SEQ ID NO:187), and the probe is StacP2 5′-CTGCGCCCGGATCCAGGC (SEQ ID NO:188).

[0035] PCR reactions were prepared in 0.5 ml thin-walled, optical grade PCR tubes (PE Applied Biosystems, Foster City Calif.) by addition of the following components: 12.5 μl of TaqMan Universal Master Mix, a 2×concentrated, proprietary mixture of AmpliTaq Gold™ DNA polymerase, AmpErase® UNG, dNTPs with UTP, passive reference dye and optimized buffer components (PE Applied Biosystems, Foster City Calif.); 2.5 μl of a mixture of forward and reverse primers (10 nM each); 2.5 μl of 400 nM TaqMan probe; 2.5 μl of 2 mg/ml bovine serum albumin (fraction V, Sigma Chemical, St. Louis, Mo.) and 5 μl of DNA template. Standard procedures for the operation of the model 7700, as described in the instrument's manual, were followed. This included the use of all default program settings with the exception of reaction volume which was changed from 50 to 25 μl. Thermal cycling conditions consisting of two min at 50° C., 10 min at 95° C., followed by 40 cycles of 15 sec at 95° C. and 1 min at 60° C. Cycle threshold (C_T) determinations, i.e. non-integer calculations of the number of cycles required for reporter dye fluorescence resulting from the synthesis of PCR products to become significantly higher than background fluorescence levels were automatically performed by the instrument for each reaction using default parameters. Assays for S. chartarum(target) sequences and G. candidum(reference) sequences in the same DNA samples were performed in separate reaction tubes.

[0036] Quantitation of S. chartarumconidia using the comparative C_Tmethod. was performed by first subtracting mean reference sequence C_Tvalues from mean target sequence C_Tvalues for both test samples and a pre-specified calibrator sample to obtain ΔC_Tvalues. Calibrator sample ΔC_Tvalues are then subtracted from ΔC_Tvalues of the test samples to obtain ΔΔC_Tvalues.

[0037] Calibrator samples were DNA extracts from mixtures of approximately 2×10⁴S. chartarum(strain UMAH 6417) and 2×10⁵G. candidumconidia. Test samples were mixed with the same quantity of G. candidumconidia prior to DNA extraction. Ratios of target sequences determined in the test and calibrator samples were then multiplied by the known quantities of S. chartarumconidia in the calibrator samples to obtain estimates of the absolute quantities of these conidia in the test samples.

[0038] Each series of DNA extracts was also analyzed using only S. chartarumtarget sequence assay results. In these calculations, calibrator sample C_Tvalues were subtracted directly from corresponding test sample C_Tvalues to obtain ΔC_T,STACvalues. These values were used in place of ΔΔC_Tvalues to determine the ratio of target sequences in the test and calibrator samples and to quantify S. chartarumconidia in the test samples as described above.

[0039] Air sampling was performed in rooms that had previously been occupied by infants diagnosed with PH from three homes in the Cleveland, Ohio area. Airborne particles were recovered in sterile BioSampler® vials (SKC Inc., Eighty Four, Pa.) connected to an AirCon-2 High Flow Sampler pump (Gilian Instrument Co., Clearwater, Fla.). Air samples were taken over an eight hour time period under passive conditions (i.e. with no activity occurring in the rooms) at a flow rate of 10 liter per min., for a total collection volume of 4.8 m³. Two additional air samples were taken in the same manner over a twelve hour period from the basement of a home in the Cincinnati, Ohio area that was also determined to contain extensive S. chartarumgrowth. One of these samples was collected under passive conditions, as described above, while the other was collected under aggressive sampling conditions (i.e. during and after a cleaning effort in the contaminated area).

[0040] Each of the BioSampler vessels was rinsed three times with 5 ml of sterile water. The pooled rinses from each vial were transferred to sterile 50 ml capped test tubes (25330-50; Corning Inc., Corning, N.Y.) and centrifuged for 15 min at 1,000×g in a Sorval RC2 centrifuge using the SS-34 rotor (DuPont Instruments, Newton, Conn.). After carefully pipeting off the top 13 to 14 ml of the supernatants, the pelleted materials in each tube were resuspended in the remaining liquid and transferred to 2 ml microfuge tubes (16-8100-27; PGC Scientific, Frederick, Md.). These suspensions were centrifuged at 14,000 rpm for 3 min in an Eppendorf micro-centrifuge (5415C; Brinkman Instruments, Westbury, N.Y.) and the majority of the supernatants were again removed by pipetting. The pellets and small amounts of liquid remaining in each tube were adjusted to either to 100 or 200 μl with sterile water.

[0041] Direct counts of putative S. chartarumconidia in 10 μl aliquots of the recovered samples were made in a hemocytometer chamber. Separate counts of up to six separate aliquots of each sample were taken over the entire grid portion of the chamber and the mean counts were converted to cell concentrations based on the instrument manufacturer's specified total volume of this portion of the chamber. Presumptive identification and scoring of particles as S. chartarumconidia were based on recognition of the characteristic size, shape and pigmentation of these conidia. Three additional 10 μl aliquots of each recovered sample were mixed with G. candidumreference conidia and subjected to total genomic DNA extraction for subsequent analysis in the model 7700 as specified above.

[0042] Yields of target sequences extracted from these conidia samples and from calibrator samples were determined from their respective C_Tresults in the model 7700 and compared using both the ΔΔC_T(including Geotrichum reference sequence data) and ΔC_T,STAC(not including reference sequence data) versions of the comparative C_Tmethod. Quantities of conidia estimated from these analyses were then compared with those determined from direct microscopic counts of the samples taken in a hemocytometer.

[0043] Results obtained by the ΔC_T,STACanalysis method and from direct counting showed good agreement for most of the samples (Example 1, FIG. 1). In thirteen of these fourteen instances, the estimate of the ΔC_T,STACmethod was within a 1-fold range of the direct counting result. The results further indicated that this level of presumed accuracy and precision (i.e. within a 50-200% range of direct counts) may be expected to occur in 95% of all analyses performed by the ΔC_T,STACmethod. Based on comparisons of results obtained by the ΔΔC_Tanalysis method for the same samples (data not shown), it was estimated that this method would provide the same level of accuracy in only about 70% of all analyses. Conidia from each of the different strains examined appeared to be quantified with similar degrees of precision and accuracy using the ΔC_T,STACanalysis method.

[0044] The sensitivity of the TaqMan assay and the functional dynamic range of the ΔC_T,STACquantitation method were further examined using ten-fold serial dilutions of S. chartarumstrain UMAH 6417 conidia stock suspensions as test samples. These samples contained expected quantities of cells that ranged from 2 to 2000, based on direct counting analyses of the starting stock suspensions. The results of these analyses were again in good agreement with the expected results (Example 1, FIG. 2). Five of the eight measurements gave estimates that coincided with the expected quantities of conidia in the samples within the relative errors of the analyses. The mean results of these analyses were within a 1-fold range of the expected values in all instances. In one of these two experiments, a low level of signal (equivalent to an estimated mean quantity of 0.27 conidia) was observed in the negative control samples. Parallel samples taken from one of the two dilution series of conidia (cf. experiment 2 in Example 1, FIG. 2) were also subjected to DNA extractions in the absence of Geotrichum cells. Although these extracts yielded slightly lower quantitative results than those obtained for the corresponding samples extracted with the normal amendment of Geotrichum cells, the difference in results was not statistically significant (p=0.35>0.05, data not shown).

[0045] A final evaluation of the TaqMan assay and ΔC_T,STACmethod was made by analyzing particulate samples collected from the inside air of four homes with known colonization by S. chartarum.TaqMan-based results were again compared with those obtained by direct microscopic observations of the samples in a hemocytometer. The two methods again gave similar mean determinations of the quantities of S. chartarumconidia in these samples with four of the five results agreeing within the relative errors of the TaqMan analyses (Example 1, Table 1). No S. chartarumconidia were found in the fifth sample by direct microscopic observation, however, this sample also appeared to approach the detection limits of the TaqMan assay with only two of the three replicate DNA extracts producing signals above background.

[0046] Example 1, Table 1. Quantification of S. chartarumconidia recovered from indoor air samples by direct microscopic counting and the ΔC_T,STACmethod as determined from TaqMan analysis.

[0047] Quantification of Fungus From Dust Using Real Time, Fluorescent Probe-Based Detection of PCR Products

[0048] Dust samples from the home of an infant with pulmonary hemosiderosis in Cleveland, Ohio (Home 1) were collected using 37-mm filter cassettes, pore size 0.8 μm, as the collection device. Samples were obtained from two rooms in the basement, the living room, and the dining room. Additional dust samples were obtained in a similar manner from the basement of a home in Cincinnati, Ohio (Home 2) containing a significant, but localized, growth of S. chartarumas determined by surface sample analysis. One sample was taken from the floor directly beneath the area of growth, a second from another location in the same room and a third from an adjacent room in the basement. All of these dust samples were sieved through a 75 μm mesh and stored in a −20° C. freezer.

[0049] Total DNAs were extracted from dust samples using glass bead milling and glass milk adsorption method. Weighed dust samples were added directly to sterile 2 ml conical bottom, screw cap tubes (506-636; PGC Scientifics, Gaithersburg, Md.), containing 0.3 g of glass beads (G-1277; Sigma, St. Louis, Mo.) and 100 and 300 μl of lysis and binding buffer, respectively from an Elu-Quik DNA Purification Kit (Schleicher and Schuell, Keene, N.H.). Ten μl aliquots of a 2×10⁷conidia/ml suspension of G. candidumin 0.5% Tween 20 were also routinely added to the tubes as a potential source of reference DNA sequences. Ten μl aliquots of S. chartarumconidia suspensions in water were also added as needed. The tubes were shaken in a mini beadbeater (Biospec Products, Bartlesville, Ohio) for one minute at a maximum speed. To bind the DNA, 25 μl of Elu-Quik glass milk suspension (Schleicher and Schuell, Keene, HN) was added to the samples and the tubes were placed on a mini-rotating mixer (Glas-Col, Terre Haute, Ind.) for 20 minutes. The samples were transferred to SPIN™ filter and catch tube assemblies (BIO 101, Vista Calif.) and centrifugation at 7500×g for 1.5 min to remove binding and lysis buffers. The retained particulates, including glass milk with adsorbed nucleic acids, were washed twice in the filter cartridges with 0.5 ml Elu-Quik wash buffer and once with 0.5 ml Elu-Quik salt reduction buffer and centrifuged as above after each wash. Nucleic acids were desorbed from the glass milk particles by two successive washes with 100 μl distilled water and collected by centrifuging the washes into clean catch tubes. Calibrator samples, used in the analytical method as standards for the quantification of S. chartarumconidia in the test samples, contained 2×10⁴S. chartarumand 2×10⁵G. candidumconidia with no dust and DNA extractions from these samples was performed in the same manner.

[0050] PCR reactions were prepared in 0.5 ml thin-walled, optical grade PCR tubes (PE Biosystems, Foster City Calif.). Each reaction contained 12.5 μl of “Universal Master Mix”- a 2×concentrated, proprietary mixture of AmpliTaq Gold™ DNA polymerase, AmpErase® UNG, dNTPs, passive reference dye and optimized buffer components. (PE Biosystems, Foster City Calif.), 0.5 μl of a mixture of forward and reverse primers at 50 mM each, 2.5 μl of 400 nM TaqMan probe (PE Biosystems, Foster City, Calif.), 2.5 μl of 2 mg/ml fraction V bovine serum albumin (Sigma Chemical, St. Louis, Mo.) and 2 μl of autoclaved water. Five μl of purified DNA extract was added to complete the 25 μl reaction mix.

[0051] The TaqMan probes and primers were obtained from the custom oligonucleotide synthesis facility at PE-Applied Biosystems (Foster City, Calif.). TaqMan probes contained a TAMRA group conjugated to their 3′-terminal nucleotide and a FAM group linked to their 5′-terminal nucleotides as the quencher and reporter fluorochromes, respectively. For Geotrichum candidum,the forward primer is NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:86), the reverse primer is GcandRl: 5′-AGAAAAMAAGTTGCCCTCTCCAGTT (SEQ ID NO:87), and the probe is GeoP2: 5′-TCAATCCGGAAGCCTCACTAAGCCATT (SEQ ID NO:88). For Stachybotrys chartarum,the forward primer is StacF4 5′-TCCCAAACCCTTATGTGAACC (SEQ ID NO:186), the reverse primer is StacR5 5′-GTTTGCCACTCAGAGAATACTGAAA (SEQ ID NO:187), and the probe is StacP2 5′-CTGCGCCCGGATCCAGGC (SEQ ID NO;188).

[0052] Standard procedures for the operation of the model 7700, as described in the instrument's manual, were followed using all of the default program settings with the exception of reaction volume which was changed from 50 to 25 μl. Thermal cycling conditions consisted of 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. Cycle threshold (C_T) determinations were automatically performed by the instrument for each assay using default parameters. Assays for S. chartarumsequences and G. candidumsequences in the same DNA samples were performed in separate reaction tubes.

[0053] To quantify conidia the mean S. chartarumcalibrator C_Tvalue was subtracted from the mean S. chartarumsequence C_Tvalues in the sample extracts to obtain ΔC_T,STACvalues. Ratios of target sequences in the test and calibrator samples were multiplied by the known quantities of S. chartarumconidia in the calibrator samples to obtain measurements of the quantities of these conidia in the test samples. similar calculations were performed in parallel using G. candidumsequence C_Tvalues from the same calibrator and test samples to determine ΔC_T,GEOvalues and quantities of these conidia in the test samples.

[0054] Then G. candidumsequence C_Tvalues were subtracted from mean S. chartarumsequence C_Tvalues for both test and calibrator sample extracts to obtain ΔC_Tvalues. Calibrator sample ΔC_Tvalues were then subtracted from the test sample ΔC_Tvalues to obtain ΔΔC_Tvalues. These values were used in place of ΔC_T,STACvalues to determine the ratios of S. chartarumtarget sequences in the test and calibrator samples and to quantify S. chartarumconidia in the test samples as indicated above.

[0055] Variances of ΔC_Twere estimated from the results of the replicate extracts of each sample by: S_ΔC²=S_Target²+S_Ref²−2rS_TargetS_ref[1], where S_Targetand S_Refare the standard deviations (SD) of the S. chartarumand G. candidumassay results, respectively, and r is the correlation coefficient between these results. Variances of ΔΔC_Twere estimated by S_ΔΔC²=S_ΔC(C)²+S_ΔC(S)²[2], where S_ΔCT(C)is given by Equation [1] applied to the calibrator results, and S_ΔCT(S), by Equation [1] applied to the test sample results. Since calibrator and test sample C_Tvalues were independent of one another; variances of ΔC_T,STACresults were estimated by: S_ΔC²=S_Calib²+S_Target²[2], where S_Calibwas the SD of the C_Tfor the calibrator. Variances of ΔC_T,GEOvalues were calculated in the same manner. Standard errors of difference were determined from the appropriate standard deviation divided by the square root of the number of replicate observations (extractions), and confidence intervals for the differences were constructed using these standard errors.

[0056] With N₀representing the number of cells in the calibrator sample, the corresponding cell numbers in test samples were estimated by N₀2^−ΔY[4], where ΔY was the estimator ΔΔC_T, ΔC_T,STAC, or ΔC_T,GEO. In this paper the term “relative error” refers the range implied by one standard deviation about ΔY, i.e. N₀2^−ΔY±S, in which S_ΔYis given by equation [2] or [3]. Confidence intervals were constructed around the estimated cell numbers by N₀2^−ΔY±t·S, where t is the appropriate Student t-value and three replicate extractions were used.

[0057] In method evaluation experiments, conidia quantities determined by the ΔΔC_T, ΔC_T,STAC, or ΔC_T,GEOmethods (N_T) were compared to “known” quantities of conidia added to the dust samples. The “known” quantities were determined from hemocytometer cell counts of three replicate aliquots (at least 400 total counts) of the conidia stock suspensions used for dilution and sample amendment. The “known” value for ΔC_T(N_H) was calculated from equation [4] based on the hemocytometer counts and dilution factors, and the differences: d=ΔC_T−known value were evaluated via analysis of variance to test the null hypothesis: d=0. The 95% confidence level range for individual observations of d was constructed, assuming d to be normally distributed, and used to characterize the precision of a single estimate utilizing TaqMan quantification. Note that when antilogs are taken, the confidence interval describes lower and upper limits to the ratio N_T/N_H.

[0058] The direct enumeration of Stachybotrys conidia in dust samples was performed by weighing dust samples, suspending them in 0.5% Tween 20 to a concentration of 1 mg/ml and, with constant mixing of the suspensions, aliquots were applied to a hemocytometer chamber. Nine replicate aliquots, or fewer if this was sufficient to enumerate at least 400 conidia, were counted in this manner for each suspension. The volumes of the examined grids were used to calculate conidia numbers per ml of suspension and these values converted to numbers per mg of dust. For comparability with relative error of the TaqMan estimates, one standard deviation ranges for direct count estimates were calculated. Conidia were assumed to be randomly distributed within each grid. Under this assumption the corresponding relative error is a range such that the observed count represents an observation one standard deviation above or one standard deviation below a Poisson variable with mean given by the lower or upper limit, respectively.

[0059] Quantitative measurements of S. chartarumconidia in dust samples taken from two contaminated homes were obtained by ΔΔC_Tanalyses of TaqMan assay results and compared with the results of presumptive direct microscopic enumeration of these conidia. Mean estimates obtained from the TaqMan assays fell within, or very close to the 0.24 to 1.04 range of direct counts that was predicted by the method evaluation experiments (Example 2, Table 1).

[0060] Analyses of known numbers of Stachybotrys conidia over a range from 2×10¹to 2×10⁴in the presence of 10 mg of composite HVAC system dust were found to provide 95% occurrence results within a range from 25% to 104% of expected values using this approach.

[0061] A second type of matrix effect that can affect PCR-based analyses of dust samples is the influence of PCR inhibitory compounds. Retention of such compounds through the DNA extraction and purification procedures occurred in only one sample in this study. A simple procedure, involving the dilution and re-analysis of a DNA extract from this sample was used to identify this matrix effect and to obtain a corrected estimate of conidia quantities. This procedure should be generally applicable so long as the concentrations of target sequences in the samples are sufficiently high to still be detectable after the inhibitor's effects are negated by dilution. In practice, however, such follow-up analyses are only likely to be necessary when significant differences are observed in the reference sequence assay results of test and calibrator samples in the initial analyses of the samples.

[0062] Evaluation of Stachybotrys chartarumin the House of an Infant with Pulmonary Hemorrhage: Quantitative Assessment Before, During and After Remediation

[0063] Air samples (Example 3, Table 1) were taken in a home under remediation for mold damage in two ways; either using a cassette filter (37 mm with 0.8 mm filter) or with a BioSampler (SKC, Eighty Four, Pa.) connecting to an AirCon-2 High Flow Sampler pump (Gilian Instrument Co., Clearwater, Fla.) calibrated at a flow rate of 10 liter per min. These samples were taken for a period between 6 and 90 hrs at 10 liter per min (L/min). During the remediation process itself, one of the workers wore a personal monitoring pump (PMP) for about 6 h a day which also used a cassette filter (37 mm with 0.8 mm filter).

[0064] Conidial stocks of the target fungus, i.e. Stachybotrys chartarum,and the reference target, i. e. Geotrichum candidum,were prepared to act as calibrator and internal standard, respectively.

[0065] Genomic DNAs were extracted from 20 μl conidial suspensions using a glass bead milling and glass milk adsorption method. Briefly, this method involved mixing test and reference conidia suspensions (10 μl ea.) with 0.3 g of acid-washed glass beads (G-1277; Sigma, St. Louis, Mo.) and 10 μl, 100 μl and 300 μl, respectively, of glass milk suspension, lysis buffer and binding buffer from an Elu-Quik DNA purification kit (Schleicher and Schuell, Keene, N.H.) in sterile 2 ml conical bottom, screw cap tubes (506-636; PGC Scientifics, Gaithersburg, Md.). The tubes were shaken in a mini beadbeater (Biospec Products, Bartlesville, Okla.) for one minute at maximum rate and DNAs were recovered in final volumes of 200 μl distilled water after performing a slight modification of the small-scale protocol provided with the Elu-Quik purification kit.

[0066] The TaqMan probes and primers were obtained from the custom oligonucleotide synthesis facility at PE-Applied Biosystems (Foster City, Calif.). TaqMan probes contained a TAMRA group conjugated to their 3′-terminal nucleotide and a FAM group linked to their 5′-terminal nucleotides as the quencher and reporter fluorochromes, respectively. For Geotrichum candidum,the forward primer is NS92F: 5′-CACCGCCCGTCGCTAC (SEQ ID NO:86), the reverse primer is GcandR1: 5′-AGAAAAGTTGCCCTCCAGTT (SEQ ID NO:87), and the probe is GeoP2: 5′-TCAATCCGGAAGCCTCACTAAGCCATT (SEQ ID NO:88). For Stachybotrys chartarum,the forward primer is StacF4 5′-TCCCAAACCCTTATGTGAACC (SEQ ID NO:186), the reverse primer is StacR5 5′-GTTTGCCACTCAGAGAATACTGAAA (SEQ ID NO:187), and the probe is StacP2 5′-CTGCGCCCGGATCCAGGC (SEQ ID NO:188).

[0067] PCR reactions were prepared in 0.5 ml thin-walled, optical grade PCR tubes (PE Applied Biosystems, Foster City Calif.) by addition of the following components: 12.5 μl of TaqMan Universal Master Mix, a 2×concentrated, proprietary mixture of AmpliTaq Gold™ DNA polymerase, AmpErase® UNG, dNTPs with UTP, passive reference dye and optimized buffer components (PE Applied Biosystems, Foster City Calif.); 2.5 μl of a mixture of forward and reverse primers (10 nM each); 2.5 μl of 400 nM TaqMan probe; 2.5 μl of 2 mg/ml bovine serum albumin (fraction V, Sigma Chemical, St. Louis, Mo.) and 5 μl of DNA template. Standard procedures for the operation of the model 7700, as described in the instrument's manual, were followed. This included the use of all default program settings with the exception of reaction volume which was changed from 50 to 25 μl. Thermal cycling conditions consisting of two min at 50° C., 10 min at 95° C., followed by 40 cycles of 15 sec at 95° C. and 1 min at 60° C. Cycle threshold (C_T) determinations, i.e. non-integer calculations of the number of cycles required for reporter dye fluorescence resulting from the synthesis of PCR products to become significantly higher than background fluorescence levels were automatically performed by the instrument for each reaction using default parameters. Assays for S. chartarum(target) sequences and G. candidum(reference) sequences in the same DNA samples were performed in separate reaction tubes.

[0068] Results of air sampling with either filters or BioSamplers indicated that the number of airborne S. chartarumspores in this PH house was low before the remediation began (Example 3, Table 1). The number of S. chartarumspores in the air, when the furnace blower was activated (typical condition for the winter months), increased by a factor of 17-47 in the living room. During demolition, the number of S. chartarumspores in the air increased by four orders of magnitude in the basement, about three orders of magnitude in the dining room and about two orders of magnitude in the upstairs bedroom (Example 3, Table 1). Thus this technology, under actual conditions, can detect the target fungus over four orders of magnitude.

[0069] Identification and Quantification of Helicobacter pylori

[0070] Culturing of Helicobacter pylorifrom environmental sources continues to be an obstacle in detecting and enumerating this organism. Selection of primer and probe sequences for the ureA gene was performed based on comparative sequence analyses of 16 strains of H. pyloriand other Helicobacter species. For Helicobacter pylorii,the forward primer is HpylF1: 5′-GGGTATTGAAGCGATGTTTCCT (SEQ ID NO:223), the reverse primer is HpylR1: 5′-GCTTTTTTGCCTTCGTTGATAGT (SEQ ID NO:224), and the probe is HpylR1: 5′-AAACTCGTAACCGTGCATACCCCTATTGAG (SEQ ID NO:225).

[0071] DNA was extracted from aliquots of ten-fold serial dilutions of H. pyloriby EluQuick kits from Schleeicher and Schuell, Inc. The cells were lysed, DNA bound to glass beads and washed with alcohol and salt reduction solutions followed by elution from filters with water. One set of extraction tubes contained only H. pylori.A second set also received 10⁷/ml E. coli.Portions of some DNA extracts were subjected to agarose gel electrophoresis and GelStar staining. Yields of high molecular weight total DNA (appearing as bands on the 1.5% gels) were estimated by comparisons of their fluorescence signals with those of a series of known mass standards (Gibco/BRL) using a model Sl fluorimager (Molecular Dynamics).

[0072] The more commonly identified non-pylori Helicobacter species were tested with H. pyloriprimers and probe (Example 4, Table 1). Results show that when compared to the negative extraction control all of these species were also negative. All obtained C_Tvalues in the range of 37 to 39. A 40 C_Tis the lowest negative value obtainable. Counts of the bacteria were high. They ranged from 10⁷to 10⁸per assay. The H. pyloristrain also was initially in this range with a C_Tvalue was 15.

[0073] Samples of serially diluted H. pyloricells spanning a 6-log concentration range were subjected to DNA extraction and TaqMan analysis.

[0074] Estimated cell quantities in the extracted samples ranged from 20 to 2×10⁶based on direct microscopic counts following staining with DAPI. Results from 5 replicate experiments showed a good correlation (r2=0.99) between TaqMan assay results (expressed as cycle threshold values) and the logarithms of expected cell numbers based on direct counts over the entire cell quantity range tested. Similar results were seen for two Helicobacter pylori strains. It was concluded that the TaqMan quantitative PCR method has the potential to provide accurate quantification of H. pyloricells in environmental samples.

[0075] Ten-fold dilutions of a single DNA extract are shown in FIG. 4 along with the corresponding regression analysis. This curve is linear all the way to a negative C_Tof 40. The R-squared is 0.999. Counts that correspond to the initial dilution can be extrapolated for the other dilutions and can be included on the X-axis. FIG. 4 shows a linear range from 1.5×10⁵to 1.5 genome equivalents.

[0076] Example 4, FIG. 4. Log (base 10)H. pyloricounts per assay are plotted against the cycle threshold values.

[0077] Conclusions

[0078] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing form the spirit and scope of the invention and without undue experimentation.

[0079] While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the intention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

[0080] Haugland, R. A. and Heckman J. L. 1998. Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum.Molec. Cellular Probes. 12:387-396.

[0081] Haugland, R. A, J. L. Heckman, and L. J. Wymer. 1999. Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR. J. Microbiol. Methods. 37:165-176.

[0082] Haugland, Vesper, Wymer. 1999. Quantitative Measurement of Stachybotrys chartarumconidia Using Real Time Detection of PCR Products with the TaqMan™ Fluorogenic Probe System. Molecular and Cellular Probes. Molecular and Cellular Probes. 13:332-340.

[0083] Vesper, Dearborn, Yike, Allan, Sobolewski, Hinkley, Jarvis, Haugland. 2000. Evaluation of Stachybotrys chartarumin the House of an Infant with Pulmonary Hemorrhage: Quantitative Assessment Before, During and After Remediation. Journal of Urban Health. 77:68-85.