Environ. Sci. Technol. 2009, 43, 6248–6255
Use of Quantitative and Conventional PCR to Assess Biodegradation of Bovine and Plant DNA during Cattle Mortality Composting W E I P I N G X U , †,‡,§ T I M R E U T E R , § YONGPING XU,† TREVOR W. ALEXANDER,§ B R A N D O N G I L R O Y E D , §,| L I J I J I N , † K I M S T A N F O R D , * ,‡ F R A N C I S J . L A R N E Y , § AND TIM A. MCALLISTER§ Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China, Alberta Agriculture and Rural Development, Agriculture Centre, Lethbridge, Alberta T1J 4V6, Canada, Agriculture and Agri-Food Canada, Lethbridge Research Centre, P.O. Box 3000, Lethbridge, Alberta T1J 4B1, Canada, and Department of Civil Engineering, University of Calgary, Calgary, Alberta T2N 1N4, Canada
Received February 4, 2009. Revised manuscript received June 18, 2009. Accepted July 6, 2009.
Understanding mortality composting requires assessing the biodegradation efficacy of carcasses and other materials of animal and plant origin. Biosecure (plastic-wrapped) compost structures were built containing 16 cattle carcasses placed on 40 cm straw and covered with 160-cm of feedlot manure. Compost was collected from depths of 80 and 160 cm (P80, P160) and DNA degradation assessed over 147 days of static composting, and during 180 days of active composting. Residual soft tissues from carcasses were collected on day 147. At P80, copies of a 171-bp bovine mitochondrial DNA (Mt171) and 138-bp plant Rubisco gene fragment (Rub138) were reduced compared to initial copy numbers (CN) by 79% and 99% after 147 days, respectively. At P160, Mt171, and Rub138 decreased compared to initial CN by 20% and 99% by day 147, respectively. After 327 days, degradation of Mt171 increased to 91% compared to initial CN. Compared to fresh tissues, residual tissues at day 147 had a 99% reduction in genomic DNA yield. Yield of DNA was related to copies of a 760bp bovine mitochondrial fragment (Mt760) which were >93% reduced at both P80 and P160 after 147 day. Secondary composting improved decomposition of bovine tissues and Mt760 was not detectable after 207 days. A 99% reduction in genomic DNA of composted tissue and >93% reduction of Mt760 suggests almost complete decomposition of carcass soft tissue after 147 days.
* Corresponding author phone: +1-403-381-5150; fax: +1-403352-4526; e-mail:
[email protected]. † Dalian University of Technology. ‡ ARD Agriculture Centre, Lethbridge. § AAFC Lethbridge Research Centre. | University of Calgary. 6248
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Introduction Mortality composting (generally a cocompost of animal carcasses, manure and straw) has become increasingly accepted for carcass disposal in the past two decades, as it is economical, reduces viable pathogens, and provides a valuable source of fertilizer for agricultural crop land (1, 2). Composting was used for disposal of poultry carcasses during an avian influenza outbreak (3, 4), but has not yet been used during an infectious disease outbreak for disposal of large carcasses, such as cattle (5, 6). The limited utilization of large animal mortality composting in disease outbreaks is partially due to uncertainty regarding pathogen inactivation and the biodegradation rate of large carcasses during biosecure composting (6, 7). Recent research demonstrated that Escherichia coli O157:H7 and Newcastle disease virus were killed after 28 days in biosecure cattle mortality compost (8). However, a better understanding is required of biodegradation rates of animal carcasses and tissues in a mortality composting environment. Mortality compost is a mix of animal and plant tissues. Conventional indicators of compost biodegradation have included dry matter, total carbon, and total nitrogen (DM, TC, TN 9-11). However, these parameters cannot distinguish between animal, plant, or microbial components within mortality compost. Consequently, degradation rates of animal tissues in the compost matrix have not been determined. In contrast, molecular techniques allow specific detection and quantification of animal, plant and microbial DNA through the use of conventional or real-time PCR assays. Since frequent turning may spread infectious agents or disturb carcass degradation, biosecure mortality compost is generally static in the primary stage (1, 2). Due to the considerable size of large mortalities, the corresponding compost structures are large, generally 1.5-2 m high (12-14). Gravimetric migration of constituents during composting may gradually increase spatial differences in biodegradation. Thus, detecting animal- and plant-specific DNA at different depths would allow spatial monitoring of the biodegradation process for material of both animal and plant origin. Degradation of mortalities was largely visually assessed in previous studies (13, 15, 16). In contrast, when decayed carcasses are no longer recognizable, molecular techniques could assess rates and levels of tissue degradation by quantifying changes in animal-specific genes. Such procedures have the potential to ensure that compost is not applied to the land before decomposition of carcasses tissues is virtually complete. To date, molecular techniques have been used to detect various pathogens from manure or compost (17-19), but limited studies have evaluated DNA of animal or plant source. Murray et al. (12) reported the presence of mitochondrial DNA for up to 190 days in transgenic swine compost, whereas Guan et al. (20) found that a target corn transgene was degraded within 14 days in compost.. The purpose of this study was to (i) determine molecular degradation of bovine carcasses after 147 days of biosecure composting and (ii) monitor biodegradation of bovine and plant source DNA over 147 days of static composting, as well as during 6 months of secondary, active composting in open windrows.
Materials and Methods Compost Construction and Sampling. Duplicate biosecure compost structures were built May 9 and 10, 2006 at Lethbridge Research Center in Alberta, Canada as described by Xu et al. (8). Briefly, 16 cattle mortalities (343 kg average 10.1021/es900310c CCC: $40.75
2009 American Chemical Society
Published on Web 07/13/2009
wt.) which had succumbed to bovine respiratory disease were placed on a 40 cm barley straw layer, and overlaid with 160 cm of beef feedlot manure. Mortalities had been previously subjected to postmortem examination and hide and rumen were punctured. The compost matrix was contained in a bunker of straw bales and lined/sealed with plastic sheeting to maintain biocontainment. Perforated air tubing was embedded within the loose straw and air vents were made on top of structures to enhance passive aeration. Final dimensions per structure were 25 × 5 m × 2.4 m with a total mass of approximately 100 tonnes. To facilitate temporal retrieval of compost samples from predetermined depths, pyramid-shaped cages, Baker Retrieval Pyramids (BRP (21)) were utilized. The BRP subjected contents to heat, moisture, microbes, and other conditions of the compost environment with minimum disturbance to the compost matrix during retrieval. Feedlot manure used to construct compost was embedded within each BRP along with samples of bovine tissues (brain, bone, and hoof) and bovine and plant material in the manure was the target in bovine- and plant-specific DNA analysis. The BRP were suspended at 80 (P80; n ) 14 per structure) and 160 cm (P160; n ) 14 per structure) depths as each structure was filled with manure. Duplicate BRP were removed at each depth from each structure at day 7, 14, 28, 56, 84, 112, and 147. Eight additional BRP were prepared but not embedded for day 0 analyses of initial DNA content. Upon removal, subsamples of compost within each BRP were obtained and stored at -40 °C prior to DNA analyses. Temperatures in BRP were monitored by type T thermocouples (Campbell Scientific, Edmonton, Canada) as described by Xu et al. (8), and averaged in quadruplicate from BRP retrieved at the same depths. After 147 days, biosecure compost structures were opened and contents were sampled at 4 m intervals. Quadruplicates of composted muscle and spinal cord residuals were collected from partially degraded carcasses. Prior to compost construction, quadruplicate fresh muscle samples were obtained from carcasses used to build compost. Tissue samples were placed on ice immediately after collection, transported to the laboratory, and stored at -40 °C prior to DNA analyses. The biosecure compost was then divided into two open windrows and composted for another 6 months before land application. Windrows were turned every 2 months (day 207, 267, and 327), and duplicate compost samples were collected from each windrow at a depth of 30 cm after turning and stored at -40 °C for further analyses. Compost DNA Extraction and Quantification. Prior to DNA extraction, subsamples of compost obtained from BRP were frozen and lyophilized for 4 days in a FreeZone FreezeDry System (Labconco Corp., Kansas City, MO), and ground through a 0.25 mm screen using a Mixer Mill MM 200 (Retsch, Haan, Germany). For each time point, duplicate compost samples from each structure at the same depth were pooled using equal amounts of DM. Subsequently, DNA was extracted from 100 mg of ground sample using QIAamp DNA stool mini kit (Qiagen Inc., Mississauga, Canada) according to manufacturer’s protocol, except that 1.7 mL ASL lysis buffer was used and DNA was eluted with 100 µL AE buffer. The extracted DNA was then visualized on a 1.0% (w/v) agarose gel (2 µL loaded) and quantified fluorometrically using QuantiT PicoGreen dsDNA Assay Kit (Invitrogen, Burlington, Canada) with a VersaFluor fluorometer (BioRad, Mississauga, Canada). Extraction and quantification were repeated in triplicate. Compost samples from open windrows were processed similarly except that samples were analyzed individually. Real-Time PCR Analyses on Compost DNA. Based on high copy numbers (CN) per cell of bovine mitochondrial DNA (Mt-DNA (22)) and plant chloroplast DNA (23), bovine
Mt-DNA (Genbank accession no. NC_006853) and plant universal chloroplast rbcL gene (encoding ribulose bisphosphate carboxylase/oxygenase, Rubisco; Genbank accession no. X04975.1) were selected as target sequences for material of animal and plant origin, respectively. A primer set framing a 171-bp bovine Mt-DNA fragment (Mt171; F: 5′-CAA TCC CTA TGG CCT CTT CA- 3′; R: 5′-AGG CTG TTT TAG GGG CAT TT-3′) was designed using Primer3 (http:// fokker. wi.mit.edu/ primer3/ input.htm); and a primer set framing a 138-bp plant Rubisco gene fragment (Rub138; F: 5′-CTT GGC AGC ATT CCG AGT A- 3′; R: 5′-CCT TTG TAA CGA TCA AGA CTG G- 3′ (24)) was utilized. In order to avoid PCR inhibitory effects caused by coextracted humic substances (25, 26), compost DNA was diluted with TE buffer (pH 8.0) to ≈10-20 ng/µL (i.e., 1:8 dilution (v/v) for day 0 compost and 1:4 dilution (v/v) for day 7-327 compost), and bovine serum albumin (BSA; New England Biolabs, Pickering, Canada) was added to each PCR (25). Each PCR mixture contained (final concentrations) 1X iQ SYBR Green Master Mix (BioRad), 1 µg/µL BSA (New England Biolabs), 0.2 µM each primer, and 1 µL of diluted compost DNA (≈10-20 ng) or standard DNA in a final volume of 25 µL. A negative control without template DNA was included in each PCR set. Standard DNA was plasmid inserted with Mt171 or Rub138 fragment, constructed using a PCR Cloningplus Kit (Qiagen), and prepared in serial dilutions from 108 to 101 copies/µL. As plasmid with inserts decreased, plasmid without inserts was substituted to keep the final plasmid concentration of each standard constant at 108 copies/µL. Standard curves were developed using 1 µL solution of Mt171 or Rub138 standard sets of each concentration. Real-time PCR amplification was performed on a Mastercycler ep realplex (4) (Eppendorf, Hamburg, Germany), using PCR conditions of 95 °C for 4 min; 45 cycles of 95 °C for 15 s, 60 °C for 1 min, and 72 °C for 15 s; and final extension at 72 °C for 5 min. Data were collected at the end of each elongation step and data analysis performed using Realplex software v.1.0 (Eppendorf). Following each PCR, 15 µL of products were visualized on 2.0% (w/v) agarose gel. Realtime PCR quantification of isolated DNA was conducted in duplicate. Detection limit for both Mt171 and Rub138 PCR was 4.60 log10 copies/g dry wt, as a minimum of 10 copies could be quantified in each reaction. Negative PCR were treated as those comprising detection limit copies of target fragments in mean and standard error calculations as well as statistical analyses. Conventional PCR Analyses on Compost DNA. To test persistence of long fragments, two sequences longer than and including Mt171 and Rub138 fragments were targeted and detected by conventional PCR. Primer sets were designed to amplify a 760-bp bovine Mt-DNA fragment (Mt760; F: 5′-CAA TCC CTA TGG CCT CTT CA- 3′; R: 5′-CCG TTT GCG TGT ATG TAT CG- 3′; Primer3) and a 822-bp plant Rubisco gene fragment (Rub822; F: 5′-CTT GGC AGC ATT CCG AGT A- 3′; R: 5′-TGC ATA CCA TGA TTT TTC TGT CT- 3′; Primer3). Conventional PCR were conducted on a Mastercycler epgradient (Eppendorf), and conditions were: 95 °C for 5 min; 50 cycles of 95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min; and final extension at 72 °C for 10 min. Each conventional PCR mixture contained (final concentrations): 1 X HotStarTaq Plus Master Mix (Qiagen), 1 µg/µL BSA (New England Biolabs), 0.2 µM each primer, 1 µL diluted DNA (≈10-20 ng) in a final volume of 25 µL. A negative control without template DNA was also included in each PCR set. Following each PCR, 15 µL of product was visualized by electrophoresis on a 2.0% (w/v) agarose gel. Positive PCR products were purified using QIAquick PCR purification kit (Qiagen) and sequenced by Macrogen USA Corp. (Rockville, MD). To exclude inhibition and false negative results caused by coextracted humic substances, PCR VOL. 43, NO. 16, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Temperature trends and DNA yields from compost at 80 (P80) and 160 (P160) cm depths over 147 days. (A) Ambient, P80 and P160 temperatures (mean + SE, n ) 4). (B) DNA yields from P80 and P160 compost over 147 days composting (mean + SE, n ) 3). The * represents differences of P < 0.05 between P80 and P160 within each time point for both A and B figures. (C, D) Gel electrophoresis of DNA extracted from P80 (C) and P160 (D) compost. Lane M: Lambda DNA/Hind III molecular size marker (Fermentas); lane 1-8: DNA isolated from compost aged at days 0, 7, 14, 28, 56, 84, 112, and 147, 2 µL loaded; lane L, GeneRuler 100bp DNA ladder plus marker (Fermentas). reactions without amplification of detectable amounts of target DNA were repeated. Absence of inhibitors were confirmed by positive target amplification after spiking standard plasmids into repeated reactions. Composted Tissue DNA Assay. For tissue samples collected from fresh (d 0) and composted (d 147) carcasses, DNA was extracted and quantified as described for ground compost samples, except that 200 mg wet samples without freeze-drying or grinding were used to extract DNA. Extracted DNA was subjected to PCR analyses as described for compost DNA with the following modifications. Real-time PCR were conducted on both Mt171 and Mt760 amplifications, and extension of 72 °C was 15 s and 1 min, respectively; 1 µL of 1:100 diluted (v/v; TE, pH 8.0) fresh tissue DNA and 1 µL of undiluted composted tissue DNA were used as PCR template, consisting of 0.1-1 ng DNA; and plasmids inserted with Mt760 fragment served as standards. Statistical Analyses. The effect of location (P80, P160) on temperatures, DNA yield, and CN of DNA fragments over 147 day composting was analyzed using Proc Mixed of SAS (27) with time treated as a repeated measure. Differences were considered significant at P < 0.05.
Results Temperature Trends and Compost DNA Yield. Compost mixture generally heated more rapidly and to higher temperatures at P80 than P160, but cooled more quickly at P80 (Figure 1A). At P80, the compost mixture reached 53.1 °C after 7 days, and stayed at 55.4-63.7 °C from day 14-56. Temperatures at P80 declined to 50.1 °C by day 84, 37.0 °C by day 112, and 16.0 °C by day 147. In contrast, at P160, temperatures peaked at 50.8 °C on day 28, but persisted at 46.2-50.8 °C during day 7-112. Temperatures declined to 35.6 °C after 147 days at P160. Temperature differences between P80 and P160 over eight time points during 147 days of composting was not significant (P ) 0.421); however, at day 14 (P < 0.001) and day 28 (P < 0.001), P80 temperatures were higher than P160; and at day 112 (P ) 0.007) and day 147 (P < 0.001), P80 temperatures were lower than those at P160 (Figure 1A). 6250
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Compost DNA yield (from BRP compost subsamples) decreased (P ) 0.003, P80; P ) 0.002, P160) after 7 days at both depths, dropping from 132.6-146.0 (d 0) to 62.6-79.8 (d 7) µg/g dry wt (Figure 1B). Thereafter, the yield of isolated DNA varied from 46.0 to 70.4 µg/g dry wt. The DNA yield did not differ between P80 and P160 over 147 days of composting (P ) 0.702), or within each collection time. DNA isolated from day 0-147 compost showed a similar pattern in molecular size from agarose gel electrophoresis, with the majority being high molecular weight DNA with minor fragmentation (Figure 1C, D). DNA yield of secondary compost averaged 53.8 ( 5.1, 47.6 ( 4.5, 42.1 ( 5.0 µg/g dry wt (n ) 4) on day 207, 267 and 327, respectively, and was not significantly lower than day 147 compost (53.0 ( 5.2 µg/g dry wt). Electrophoresis of DNA from secondary compost showed a similar pattern as that of day 147, with the majority of DNA being high molecular weight with little fragmentation (data not shown). Bovine and Plant DNA Degradation in Compost. On the basis of quantitative and conventional PCR analyses, bovine Mt-DNA degraded more rapidly at P80 than in close proximity to the carcasses at P160 (Figure 2). At P160, CN of Mt171 increased until day 84, while at P80, the CN were 5.82 log10 copies/g dry wt on day 0, 14, and 28, and decreased to 5.64 log10 copies/g dry wt after 56 days, a 28% reduction compared to day 0 compost (P ) 0.028). Thereafter, Mt171 was continuously degraded and reached the minimum of 5.10 log10 copies/g dry wt on day 147, a 79% reduction from day 0 (P < 0.001). The long fragment, Mt760, was not detectable by conventional PCR after 112 days at P80 (Figure 2A, B, E). In contrast, at P160 both Mt171 and Mt760 were detected over 147 days of composting and concentrations of both increased from day 7-112. At P160, copies of Mt171 increased 97% after 7 days composting (P < 0.001), rising from 5.76 to 6.08 log10 copies/g dry wt. Elevated copies of Mt171 were sustained from day 7-112, varying from 6.08 to 6.60 log10 copies/g dry wt, representing a 97-584% increase from day 0. However, after 147 day Mt171 declined to 5.69 log10 copies/g dry wt, a 20% reduction from day 0 (P ) 0.472). Similarly to Mt171, Mt760 accumulated from day 7-112 at P160, but was
FIGURE 2. Real-time and conventional PCR analyses on persistence of 171-bp (Mt171) and 760-bp (Mt760) bovine Mt-DNA fragments at 80 (P80) and 160 (P160) cm depths over 147 days composting. (A-D) Gel electrophoresis of Mt171 real-time and Mt760 conventional PCR products. Lane M: pUC8 size Marker (Fermentas); lane 1-8, compost aged 0, 7, 14, 28, 56, 84, 112, 147 days; lane N, negative control. (E) Real-time PCR analysis of copy numbers of Mt171(mean ( SE, n ) 6). The * represents P < 0.05 between P80 and P160 compost within each time point. reduced after 147 days compared to day 0 based on conventional PCR signal (Figure 2C, D, E). Sequencing analyses of Mt760 showed >99% identity of amplified sequences to the reference bovine gene. Concentrations of Mt171 at P80 were lower than P160 over 147 days (P < 0.001), even within each time point from day 7-147. In contrast to bovine Mt-DNA, plant Rubisco DNA degraded more rapidly over 147 days at both depths (Figure 3). At P80 and P160, CN of Rub138 decreased from 7.52-7.63 of day 0, to 5.74-5.82 by day 28, and 5.33-5.49 log10 copies/g dry wt by day 147, representing a 98% (P < 0.001) and 99% (P < 0.001) reduction from day 0, respectively (Figure 3A, C, E). Long fragment of Rub822 was not detectable by conventional PCR after 28 days at both depths (Figure 3B and D). Sequencing analyses confirmed a > 95% identity of the amplified Rub822 to the reference plant gene. CN of Rub138 between P80 and P160 over 147 days differed significantly (P ) 0.025, P80 > P160), as well as on day 7 (P < 0.001, P80 > P160), day 14 (P < 0.001, P80 > P160), and day 84 (P ) 0.001, P80 < P160). Both long fragments Mt760 and Rub822 were not detectable in any of the DNA extracted from secondary compost (data not shown). For short bovine fragment Mt171, 5.2 ( 0.1, 4.9 ( 0.2, and 4.7 ( 0.1 log10 copies/g dry wt were detected, and for plant Rub138, 5.5 ( 0.1, 5.3 ( 0.3, and 5.2 ( 0.2 log10 copies/g dry wt were detected on day 207, 267, and 327, respectively (Figure 4). CN of Mt171 on day 207, 267, and 327 were significantly lower than on day 0, whereas day 267 (P ) 0.019) and day 327 (P ) 0.003) were also lower than day 147. After 327 day of combined static and active composting, there was a 91% reduction in bovine Mt171 and 99% reduction in plant Rub138 (Figure 4C and D). Bovine DNA Degradation in Composted Tissue. DNA yields from fresh bovine muscle (day 0), composted bovine muscle and spinal cord (day 147) were 65.7, 0.22, and 0.22 µg/g wet wt, respectively (Figure 5). Corresponding DNA CN
of fresh muscle, composted muscle and spinal cord were 9.28, 8.41, and 7.68 log10 copies/g wet wt for Mt171, and 9.13, 7.93, and 7.35 log10 copies/g wet wt for Mt760 (Figure 5). Compared to fresh tissue, there was a > 99% reduction in DNA yield, > 85% degradation in Mt171 copies, and >93% degradation in Mt760 copies of composted tissues after 147 days static composting. DNA yield of fresh muscle was higher than that of composted muscle and spinal cord (P < 0.001), while there was no difference between composted muscle and spinal cord (P ) 0.992). CN of both Mt171 and Mt760 were ranked as fresh > composted muscle (P < 0.001) > composted spinal cord (P < 0.001).
Discussion DNA as an Indicator of the Biodegradation Process. Conventional compost measurements such as TC, and TN (9-11) characterize the chemical nature of compost, but do not provide information on the decomposition of specific constituents, such as plant and animal tissues. In a companion study, TC generally decreased over 147 days at P80 and P160 (8), but the rate and extent of decomposition of bovine tissues could not be easily defined, as TC only declined by 20%. In contrast, use of bovine-specific DNA-based procedures enabled us to document that 79% and 20% of bovine Mt171 were degraded at P80 and P160 after 147 days, respectively. As P160 was in close proximity to the carcass layer, the increased copy number of Mt171 detected at P160 over the first 84 days of composting was likely related to release of DNA from carcass degradation and diffusion into the surrounding compost. Moisture content at P160 was significantly higher than P80 over 147 day (8), indicating downward movement of moisture. It is also possible that bovine DNA was concentrated in the lower regions due to gravimetric flow within the compost, similar to movement of free DNA with water through soil profiles (28). However, VOL. 43, NO. 16, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Real-time and conventional PCR analyses on persistence of 138-bp (Rub138) and 822-bp (Rub822) plant Rubisco gene fragments at 80 (P80) and 160 (P160) cm depths over 147 days of composting. (A-D) Gel electrophoresis of Rub138 real-time and Rub822 conventional PCR products. Lane M: pUC8 size Marker (Fermentas); lane 1-8, compost aged 0, 7, 14, 28, 56, 84, 112, 147 days; lane N, negative control. (E) Real-time PCR analysis of copy numbers of Rub138 (mean ( SE, n ) 6). The * represents P < 0.05 between P80 and P160 compost within each time point.
FIGURE 4. Real-time PCR analyses on reduction of 171-bp (Mt171) bovine Mt-DNA and 138-bp (Rub138) plant Rubisco gene fragments during secondary composting. (A, B) Gel electrophoresis of Mt171 and Rub138 real-time PCR products of the secondary compost. Lane M: pUC8 size Marker (Fermentas); Lane N and P: negative and positive control; Lane 1-4: samples collected on days 207, 267, and 327. (C, D) Real-time PCR quantification of Mt171 and Rub138 in static compost on day 0 and 147 (mean + SE, n ) 6), and secondary compost on days 207, 267, and 327 (mean + SE, n ) 4). Values indicate % reduction compared to day 0. concentration of Mt-DNA after 147 days of composting was only 20% lower than that present on day 0 at P160. These results suggest that significant quantities of bovine Mt-DNA remained in lower regions of the compost and that accumulation of moisture, as opposed to nutrient limitations, restricted microbial growth and heat production. Secondary 6252
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open windrow composting further improved degradation of bovine Mt-DNA, as long fragment Mt760 was not detectable on days 207, 267, or 327, and decomposition of short fragment Mt171 reached 91% after 327 days of composting. A similar observation that animal-specific DNA was present after static composting but not secondary composting occurred in swine
FIGURE 5. Bovine carcass decomposition evaluated by DNA yield, copy numbers of 171-bp (Mt171) and 760-bp (Mt760) bovine Mt-DNA in fresh muscle (day 0) and composted muscle and spinal cord (day 147). (A) Genomic DNA yields from fresh and composted bovine tissues (mean + SE, n ) 4). (B) Real-time PCR quantification of Mt171 and Mt760 in fresh and composted tissues (mean + SE, n ) 8). Values indicate % reduction of composted tissue from fresh tissue on day 0. mortality compost (12), where a ≈500-bp swine Mt-DNA was detectable after 100 days of static composting, but could not be amplified after another 300 days in secondary compost turned every 3-4 mo. In contrast to bovine Mt-DNA, 98% of plant Rub138 was degraded at both depths after 28 d. This suggests that DNA in undigested feed and bedding (cereal straw) is rapidly degraded and that plant cell contents are likely primary substrate for microbes during early stages of composting. Similarly, Guan et al. (20) reported that a transgenic fragment in genetically modified corn was no longer detectable after 14 days composting. Perhaps these results are not surprising given that there are only five copies of this trangene per genome (29), but our study confirmed a similar rapid degradation even when a gene with 500-50 000 copies per cell was targeted (23) as the Rubisco gene quickly declined to negligible levels. However, there was evidence that microbial activity was not uniform throughout the compost as one region of P160 only achieved temperatures of 17.6-30.5 °C compared to the average P160 temperature of 45.0-47.0 °C during days 14-45. Levels of Rub138 at the lowtemperature location remained higher, indicated by the relatively strong PCR signal of P160 compost on day 84. Consequently, rate of decomposition of DNA and organic matter was spatially variable within compost. Compost DNA Detection and Quantification. Bovine MtDNA has been widely used as an indicator of bovine tissue in environmental and food studies (30), due to high CN per cell, ranging from 220 to 1720 (22) and high degree of specificity (31, 32). Compared to low copy genes, such as transgenes or growth hormone gene which have
