Journal of Microbiological Methods 75 (2008) 432–436

Contents lists available at ScienceDirect

Journal of Microbiological Methods

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h

An effective method for isolation of DNA from pig faeces

and comparison of five different methods
Jun-ni Tang, Zhi-guang Zeng, Hong-ning Wang ⁎, Tai Yang, Peng-ju Zhang, Yu-ling Li, An-yun Zhang,
Wen-qiao Fan, Yi Zhang, Xin Yang, Su-jun Zhao, Guo-bao Tian, Li-kou Zou
Bioengineering Research Center for Animal Disease Prevention and Control, School of Life Science, Sichuan University, Chengdu 610064, China

a r t i c l e i n f o a b s t r a c t

Article history: Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor
Received 2 July 2008 recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for
Received in revised form 20 July 2008 extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment
Accepted 20 July 2008
with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and β-mercaptoethanol,
Available online 23 July 2008
followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a
Keywords:
1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA
Pig faeces extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain
DNA extraction reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method
β-mercaptoethanol represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens
Comparison and was notably better than using the QIAamp® DNA Stool Mini Kit.
PCR © 2008 Elsevier B.V. All rights reserved.

1. Introduction
The intestinal tract of animals harbors a large and complex community
of microbes which possess of at least 400–500 different microbial species,
constituting a complex ecosystem (Yang et al., 2008). Faecal samples are
increasingly used as sources of DNA for genetic and ecological research,
which contain intestinal bacteria that may provide useful information
concerning gastrointestinal tract health for animals (Zhang et al., 2006). In
recent years, molecular methods such as PCR-based techniques have also
been used to analyze dominant bacterial groups of the animal gut
microbiota (Thiel and Blaut, 2005). These studies have generally not relied
on microbial cultivation, but have been carried out using the specific
genome DNA of bacterial groups or species of interest from faecal samples.
Such DNA can reveal the complexity of microbial communities living in
these environments through PCR amplification of the 16S ribosomal RNA
(16S rRNA) gene sequence and other molecular phylogenetic techniques
(Barton et al., 2006). To facilitate the study of intestinal microbial com-
munities, several DNA extraction methods have been reported for use
with faecal samples (Ward and Wang, 2001; Subrungruang et al., 2004;
Thornton and Passen, 2004; Nantavisai et al., 2007). Almost all of them are
difficult and time consuming, dealing with high amount of toxic materials
like phenol. Their results also vary due to sample collection method and
further purification treatment (Abbaszadegan et al., 2007). Moreover,
studies have shown that organic samples, such as faeces, contain
⁎ Corresponding author. Tel./fax: +86 28 8547 1599.
E-mail address: Whongning@163.com (H. Wang).
“inhibitors” which can reduce PCR sensitivity by 1000-fold (Ward and
Wang, 2001; Harris and Barletta, 2001). The other problems are often
encountered in terms of relatively low DNA yields (Yu and Morrison,
2004). Therefore, extracting DNA from organically complex samples is
technically challenging.
Generally, methods for extracting DNA from faecal samples
previously based on culture enrichment, or extraction of nucleic
acids using phenol, ion-exchange chromatography, silica, or combina-
tions (Thornton and Passion, 2004). In the present feasibility study, we
developed a new effective, simple and combined protocol that could
be used to isolate PCR-quality DNA from pig faeces adaptable to detect
16S rDNA fragments and other molecular techniques.
2. Materials and methods
2.1. Collection and preparation of faecal samples
Fresh faecal samples were obtained from healthy multiparous
sows of Rongchang pig. The samples were collected in sterile bags and
kept on ice, immediately taken to the laboratory and subsequently
frozen at −20 °C or −70 °C.
2.2. Pretreatment of samples
Two different ways of the pretreatment were conducted. The effect
of the pretreatment methods was evaluated by using method (M) (see
Section 2.4) with the same subsamples.

0167-7012/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2008.07.014
 J. Tang et al. / Journal of Microbiological Methods 75 (2008) 432–436
Fig. 1. Average amounts of DNA yield (A) and 1.0% agarose gel image displaying genomic DNA extracted from the pig faecal samples by using various methods run with λ DNA cut with
HindIII (DNA marker) (B). A: Labels are of the form X–Y, where X is the DNA extraction method and Y is the extracted average total DNA yield [μg/g (wet weight)]. DNA was measured
by using a SmartSpec™ plus (Bio-Rad, USA). B: Lanes 1–3: M1; Lanes 4–6: M2; Lanes 7–9: M3 (kit); Lanes 10–12: M4; Lanes 13–15: M (new method).
2.2.1. Treatment A
The frozen samples (1.6 g) were transferred to a sterile tube (50 ml)
adding 30 ml of ice-cold 0.05 M phosphate-buffered saline (PBS)
(150 mM NaCl, 10 mM Na2HPO4, 20 mM NaH2PO4, pH 7.4), vortexed
fully. Samples were centrifuged at 500 ×g for 4 min, and the su-
pernatants were transferred to new 50-ml sterile tube. Pellets were
resuspended in 30 ml PBS buffer, mixed thoroughly and centrifuged at
500 ×g for 4 min. Repeat the procedure two times. All the supernatants
were centrifuged at 9000 ×g for 5 min. The cell pellet was harvested and
resuspended in the mixture of 4 ml of TE buffer (pH 8.0), and stored at
−20 °C for use.
2.2.2. Treatment B
The pretreatment method was described by Thiel and Blaut (2005)
previously with some modification. Briefly, the frozen samples (1.6 g)
were transferred to a sterile tube (50 ml), diluted in 10 ml of ice-cold
0.05 M PBS buffer (150 mM NaCl, 10 mM Na2HPO4, 20 mM NaH2PO4,
pH 7.4) and homogenized by the addition of ten sterile glass beads
(3 mm in diameter), vortexed fully for 3 min. Subsequently, the sam-
ples were centrifuged at 400 ×g for 2 min to remove glass beads and
larger particles. The larger particles were resuspended in 3 ml of ice-
cold PBS buffer, mixed thoroughly and centrifuged at 400 ×g for 2 min
again. All the suspension was transferred to a new 50-ml sterile tube
and mixed with 3 vol of 4% polyformaldehyde, incubated on ice for 1 h.
Following fixation, the cell suspension was centrifuged at 8000 ×g
for 3 min and the cell pellet was resuspended in 4 ml PBS buffer,
mixed with 4 ml absolute ethanol and stored at −20 °C for 20 min.
Subsequently, the cell pellet was harvested by centrifugation at
8000 ×g for 3 min and resuspended in 4 ml of TE buffer (pH 8.0) for
use.
2.3. Optimization of the buffer components
Optimization of β-mercaptoethanol, PVP, and NaCl concentra-
tions was carried out in this assay. The optimized concentrations of
β-mercaptoethanol (0%, 1%, 2%, 3%, 4%), and PVP (0.2%, 0.5%, 1%, 2%),
and NaCl (1.4 M, and 2.0 M) in the lysis buffer were tested to extract
DNA by using total DNA yield and gel analysis.
2.4. DNA isolation methods
Genomic DNA was extracted from 0.2 g (wet weight) pig faeces for
each tube by using five different methods with all extraction
techniques being conducted on a subsample of the same homogenized
initial sample. M, M1, and M2 were based on the pretreatment of
samples conducted with treatment B (described in Section 2.2.2). M3
(kit), and M4 applied the faecal sample directly. All the extractions
were performed in triplicates.
433
Method (M) was a new method for extracting DNA from pig faecal
samples in this study.
The detailed extraction protocol was as follows:
1) Transfer each aliquots of 500 μl pretreatment sample into a new
2.0 ml reaction tube;
2) Harvest cells by centrifugation at 16,000 ×g for 10 min;
3) Add 1 ml of hot CTAB extraction buffer (pre-heated at 65 °C water
bath) [100 mM Tris–HCl (pH 8.0), 2.0 M NaCl, 20 mM EDTA, 2%
CTAB], vortex fully;
4) Add 1% PVP and vortex fully;
5) Then add 2% β-mercaptoethanol into each tube and vortex fully;
6) Incubate the tubes for 1 h (or 2 h) in a 65 °C water bath with
occasional shaking (~15 min);
7) Centrifuge at 16,000 ×g for 10 min;
8) Transfer the supernatant into a new 2.0 ml reaction tube;
9) Add 1 vol of chloroform: isoamyl alcohol (24:1, v/v);
10) Centrifuge at 16,000 ×g for 10 min;
11) Transfer the supernatant into a new 2.0 ml reaction tube;
12) Add 1 vol, equal to the transferred supernatant, of chloroform;
13) Centrifuge for 10 min at 16,000 ×g and 4 °C;
14) Transfer the supernatant into a new 2.0 ml reaction tube;
15) Add 2 vol of ice-cold absolute ethanol;
16) Incubate at −20 °C for 30 min;
17) Centrifuge for 10 min at 16,000 ×g;
18) Remove the supernatant completely using a pipette;
19) Wash DNA pellet with ice-cold 70% ethanol;
20) Air dried;
21) Resuspend DNA pellet in 50 μl of sterile ultrafiltered water with
RNase (20 μg/ml);
22) Incubate the tube at 37 °C for 30 min;
23) Store DNA at −20 °C before use.
Method 1 (M1) was the chemical-enzymatic method of Niemi et al.
(2001). Each aliquot of 500 μl pretreatment sample was transferred
into a new 2.0-ml tube. Then cells were disrupted with 25 μl lysozyme
(20 mg/ml) in 37 °C for 30 min. Subsequently, the tube was added with
50 μl 10% sodium dodecyl sulfate (SDS), and 15 μl proteinase K (10 mg/
ml) in slightly alkaline TE buffer incubated in a 55 °C water bath for
2 h. DNA was extracted with phenol:chloroform:isoamyl alcohol
(25:24:1, v/v/v) and precipitated with isopropanol. Finally, the DNA
pellet was suspended in 50 μl of sterile ultrafiltered water.
Method 2 (M2) was described by Tang et al. (2006) previously.
Briefly, the pellet was harvested and suspended in 1 ml of ice-cold 70%
ethanol on ice for 20 min. The cells were collected and resuspended in
the mixture of 467 μl of Tris–EDTA (TE) (10-mM Tris–Cl, 1-mM EDTA,
pH 8.0), 12 μl of 20-mg/ml lysozyme and 1 μl of 1-mg/ml ribonuclease
(RNase), and incubated on ice for an additional 1 h. The tubes were
434 J. Tang et al. / Journal of Microbiological Methods 75 (2008) 432–436

2.5. Polymerase chain reaction (PCR)

Following the DNA extraction, amplifications by PCR were performed

from each method with 16S rDNA specific primers [forward primer 27F
(5′-AGAGTTTGA TCC TGG CTC AG-3′) and the universal reverse primer
1492R (5′-GGCTTACCTTGTTACGACTT-3′)] (Hayashi et al., 2002). Reac-
tions were carried out in a volume of 20 μl with a 10× PCR buffer, 2 μl;
2.0 mM MgCl2; 200 μM of each nucleotides dATP, dTTP, dGTP and dCTP; 1
1 U of Taq polymerase (TIANGEN), 1 μl of purified sample DNA and with
primers 0.5 μM. The PCR was carried out for 30 cycles at 95 °C for 30 s,
59 °C for 30 s, and 72 °C for 1 min 30 s, with a first cycle of 5 min at 95 °C
and a final cycle of 8 min at 72 °C. Amplifications were performed for a
minimum of three times for each sample, and a negative control (water)
was carried out.

2.6. Statistical analysis

Statistical analyses were performed by using GraphPad PRISM®

software.

3. Results

3.1. Comparison of pretreatment

Two different pretreatment methods were compared. Sample pre-

paration with 4% polyformaldehyde could improve the DNA yields
effectively. The average DNA yield ratio of treatment B and treatment
A was close to 1.6-fold followed by using method (M).
Fig. 2. Average amounts of DNA yield extracted from different concentrations of β-
mercaptoethanol (0%, 1%, 2%, 3%, and 4%) (A) and agarose gel analysis of the extracted
3.2. DNA yield and purity
DNA run with λ DNA cut with HindIII (DNA marker) (B).

Total amounts of DNA yield extracted with five extraction methods

then incubated in a 68 °C water bath, following the addition of 53 μl of varied considerably, with the new method (M) from which the highest
10% sodium dodecyl sulfate (SDS), 87 μl of 5-M NaCl and 69 μl of yields could be obtained being significantly different from others. The
cetyltrimethylammonium bromide (CTAB)/NaCl solution (1% CTAB,
0.73-M NaCl). Subsequently, DNA was extracted with phenol:chlo-
roform:isoamyl alcohol (25:24:1, v/v/v) and precipitated with ethanol.
The DNA pellet was suspended finally in 50 μl of sterile ultrafiltered
water and stored at −20 °C before use.
Method 3 (M3) was conducted by using QIAamp® DNA Stool Mini Kit
(Purchased from QIAGEN) according to the manufacturer's instructions.
Method 4 (M4) was described by Rousselon et al. (2004) previously.
Briefly, faeces (0.2 g/sample) were suspended in 100 μl of 4 M guanidine
thiocyanate −0.1 M Tris (pH 7.5), 30 μl of 10% N-lauroyl sarcosine, and
500 μl 0.1 M phosphate buffer (pH 7.4). Then the tube was incubated at
70 °C for 1 h. 500 μl of 0.1-mm-diameter silica beads (Sigma), previously
sterilized by autoclaving, was added and the tube was shaken for 10 min.
PVP (15 mg) was added to the tube, which was vortexed and centrifuged
for 3 min at 12,000 ×g. After recovery of the supernatant into a 2.0-ml
tube, the pellet was washed with 500 μl of lysis buffer [50 mM Tris (pH
8.0), 20 mM EDTA (pH 8.0), 100 mM NaCl, 1% PVP] then centrifuged for
3 min at 12,000 ×g, and the supernatant was added to the first
supernatant in the same 2.0-ml tube. The washing step was repeated
three times. The pooled supernatant (about 2 ml) was briefly centrifuged
to remove particles and then split into two 2.0-ml tubes. Nucleic acids
were precipitated by the addition of 1 vol of isopropanol for 10 min at
room temperature and centrifuged for 15 min at 20,000 ×g. Pellets were
resuspended and pooled in 100 μl of sterile ultrafiltered water with
RNase (20 μg/ml), and incubated at 37 °C for 10 min.
All extractions were performed in triplicates and eluted in 50 μl of
sterile ultrafiltered water. The purity of DNA was assessed spectrophoto-
metrically by calculating A260/A280 ratios and the yield of DNA was calcu-
lated from the A260 value by using a SmartSpec™ plus (Bio-Rad, USA). The
quality of DNA extracted from each method was evaluated by running 5 μl Fig. 3. Average amounts of DNA yield extracted from different concentrations of PVP
of purified DNA on a 1.0% agarose gel using λ DNA cut with HindIII as DNA (0.2%, 0.5%, 1%, and 2%) (A) and agarose gel analysis of the extracted DNA run with λ
marker (TIANGEN). The presence of PCR inhibitors was analyzed by PCR. DNA cut with HindIII (DNA marker) (B).
 J. Tang et al. / Journal of Microbiological Methods 75 (2008) 432–436 435

Fig. 4. PCR amplification of 16S rDNA from extracted DNA of different methods. Gel electrophoresis showing PCR products (1560-bp) following amplification of 1 μl of each DNA
template per reaction tube from different methods. Each method was examined at three separate occasions. Aliquots (8 μl) of PCR products were loaded onto 1% agarose gel, subjected
to electrophoresis for 40 min at 100 V in 1× TAE buffer and visualized with UV illumination. Lanes 1–3: PCR products from M1; Lanes 4–6: PCR from M2; Lanes 7–9: PCR products from
M3 (kit); Lanes 10–12: PCR products from M4; Lanes 13–15: PCR products from M; Lane C: negative control.

average yields for M, M1, M2, M3 and M4 were 163.5±7.34; 124.4±15.33;
82.4±11.03; 14.3±1.13; and 69.9±5.15 μg/g (wet weight) (Fig. 1A). Method
(M) accounts for the top 1, resulting in a 1.3- to 11-fold increase in average
DNA yield compared to M1, M2, M3 and M4. The M3 (kit) method
consistently gave lower yields than the other methods. The A260/A280
ratio, indicating contamination by proteins, is about 2.0 for pure DNA
and should exceed 1.7 for nearly pure nucleic acid extractions from
environmental samples. In our study, the A260/A280 ratios for M, M1, M2,
M3, and M4 were 1.82±0.014; 1.80±0.030; 1.67±0.010; 1.84±0.010; and
1.77±0.017 respectively. With respect to DNA yield and purity in this
study, method (M) was the best DNA extraction method for pig faeces.
3.3. Evaluation of buffer components and incubation time
We prepared a series of extraction buffers and concentration to
estimate the influence of various reagents in the buffer on successful
DNA extraction. Fig. 2 shows examples of the extracted DNA prepared
by using different β-mercaptoethanol buffers (0%, 1%, 2%, 3%, 4%). The
DNA agarose gel suggested that the presence of β-mercaptoethanol in
the extraction buffer was essential, and its omission resulted in un-
successful DNA extraction with our procedure (Fig. 2B). Use of 1%, 2%,
and 3% β-mercaptoethanol led to more effective extraction than did
use of 4% (Fig. 2A). Fig. 3 shows examples of the extracted DNA pre-
pared by using different PVP buffers (0.2%, 0.5%, 1%, 2%). Use of different
PVP buffers all led to effective extraction (Fig. 3B). However, use of 1%
PVP has a higher DNA yield than did use of 0.2%, 0.5%, and 2% (Fig. 3A).
The addition of NaCl was likely to have some advantage in the ex-
traction buffer. However, 1.4 M and 2.0 M NaCl have no statistically
significant difference. Incubation time of 1 h or 2 h in a 65 °C water bath
after adding CTAB lysis buffer was also found to have no significant
difference to the yield of DNA. Therefore, the subsequent extraction of
DNA applied 1 h incubation in 65 °C water bath to shorten the time.
3.4. Evaluation of DNA extraction methods
Five different DNA extraction methods were examined to ascertain
their relative effectiveness, Some of which could extract total DNA
successfully from pig faeces, as demonstrated by agarose gel detection
and successful 16S rDNA-specific PCR (Figs. 1B and 4). Of the five
methods, M was the most effective method for the extraction of
DNA prepared from the pretreatment samples. M1, M2, and M4 were
also effective in extracting DNA from faeces. M3 was a commercial kit
(QIAamp®) method. The DNA extracted by the kit method could not be
detected on agarose gel. However, successful PCR outcome could be
observed from faecal samples in all five methods (Figs. 1B and 4). In the
study, repetition of testing DNA extract with method (M) provided
reproducible results which confirmed the superiority of this method,
with consistently higher levels of DNA yield and PCR product from each
faecal sample.
4. Discussion
The isolation of DNA is one of the most commonly used procedures
in genetics, molecular biology and biochemistry. To date, numerous
methods have already been devised to isolate genomic DNA from
various biomaterials, depending on the sources used (Ki et al., 2007).
Faeces are a very complex mixture of biological and non-biological
elements that can significantly inhibit PCR reaction (Iacovacci et al.,
2003). Very few methods for preparing DNA template from pig faecal
samples were developed. In this study, we developed a simple, effective,
and inexpensive method for DNA extraction from pig faeces by com-
bining some important factors of existing protocols (Lee et al., 2003;
Rousselon et al., 2004; Rouhibakhsh et al., 2008). Our procedure was
tested by repeating the extraction, which has shown good stability and
repetitiveness. This method allowed us to obtain the high quality mole-
cular weight DNA template from pig faeces samples. Our procedure
contains four major steps: (1) treating the faecal samples by 4% poly-
formaldehyde; (2) adding 1% PVP; (3) adding 2% β-mercaptoethanol
in CTAB lysis buffer; and (4) extracting DNA by using chloroform (no
phenol). All four steps were proved to be essential for obtaining DNA of
good quality and relatively large quantity. Our procedure was developed
primarily for isolation of DNA from freshly collected pig faecal sample or
sample stored at −70 °C for up to a few days. Later tests proved that with
this method DNA could also be isolated from the sample stored at −20 °C
for up to 1 or 2 months. Thus, the new combined method can be po-
tentially useful for DNA extraction from all faecal samples. This might
have great potential for the study of microbial communities concerning
gastrointestinal tract health for all animals.
Every type of sample, because of its own nature, requires optimiza-
tion of the extraction methods (Roose-Amsaleg et al., 2001). The faecal
sample contains variable concentrations of inhibitory substances such
as biliary salts, proteins, polysaccharide complexes, bilirubins and phe-
nolic compounds, depending on diet, intestinal flora and health con-
ditions of the host (Gonçalves et al., 2008). In designing this protocol, we
investigated several factors influencing the release of nucleic acid from
the sample. CTAB is included in extraction buffers for DNA as reagents
for protein denaturation. β-mercaptoethanol is a strong reducing rea-
gent and is included in DNA extraction buffers to prevent oxidation of
436 J. Tang et al. / Journal of Microbiological Methods 75 (2008) 432–436
polyphenols present in the faeces extract. Introduction of PVP that
assures efficient removal of inhibitors that may coprecipitate with DNA
during extraction after treatment with a lysis solution, improved the
final amplification. In this study, the concentration of the important
factors (β-mercaptoethanol, and PVP) was optimized. The concentration
of β-mercaptoethanol was initially presumed 0–10% and the concentra-
tion of PVP was 0–4% in our preliminary studies. The ultimate range was
reduced gradually according to extraction yields (for β-mercaptoethanol
0–4%; for PVP 0–2%). The adding optimum of β-mercaptoethanol and
PVP was further confirmed by total DNA yield and gel analysis. This
optimized technique would be suitable for other complex matrices, at
least faeces from other domestic species. The procedure is quite simple
and rapid, and does not require expensive reagents (proteinase K and
lysozyme) and hazardous chemical reagents (phenol). Therefore, an easy
and rapid method was optimized for isolating the pig faecal DNA with
reduced PCR inhibitors.
Moreover, DNA yields were found to vary depending on the
method used. In this study, we compared the efficiency of our method
with other procedures published previously and QIAamp® DNA Stool
Mini Kit. The methods developed to date for extracting microbial DNA
are of two types: cell extraction methods and direct lysis methods
(Roose-Amsaleg et al., 2001; Herrera et al., 2007). Cell extraction de-
pends on the isolation of microbial cells from their environmental
matrix, prior to lysis to release DNA. These methods use successive
cycles of blending and differential centrifugation to recover the intact
microbial cells. Direct lysis method does not require cell isolation,
including direct cell lysis in the sample after which DNA is extracted.
In the study, our improved method (M), M1, and M2 applied the first
approach, M3 (kit) and M4 applied the second approach. The first
approach was slightly superior to the direct approach in terms of DNA
yield and PCR amplification in our research. QIAamp® DNA Stool Mini
Kit was used as a control for extraction and purity of DNA. Though this
kit has been proved to be effective at overcoming problems of co-
extracted PCR inhibitors from faeces samples (Foltan, et al., 2005;
Nechvatal et al., 2008), and the PCR amplification bands of 16S rDNA
was also successfully obtained in our study, a potential problem with
this kit was the low efficiency of DNA yield.
In conclusion, the goal of the study was to simplify processing of
faecal specimens and develop a protocol that was amenable to large-
scale manipulation for PCR analyses with efficient extraction of highly
pure DNA. Depending on time, costs, and reproducibility, our method
was demonstrated to be an effective DNA extraction with consistently
higher levels of DNA yield and relatively inhibitor free from pig faecal
specimens. Therefore, our new extraction method could provide a
reference source for other researchers to extract DNA from faecal
samples in a simple format.
Acknowledgements
This research was supported by the National “11th Five-Year Plan”
Science and Technology Supporting Programs (2007BAD51B05;
2006BAD14B05-5; 2006BAK02A19) and China Postdoctoral Science
Foundation funded project. The authors wish to thank Fu Li-zhi, Zhai
Shao-qin, and Fu Wen-gui from Chongqing Academy of Animal Sciences
for their assistance in providing the Rongchang pigs.
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