全 文 ：农业生物技术学报，2011年，第 19卷，第 5期，第 973~980页
Journal of Agricultural Biotechnology, 2011, Vol.19, No.5, 973~980
DOI: 10.3969/j.issn.1674-7968.2011.05.026
基金项目：本研究由国家科技支撑计划(No.2011BAK10B02)资助
收稿日期：2010-12-07 接受日期：2011-02-24
技术改进
Updated Technology
Double-colored Real-time Fluorescence PCR Method for Detection of
Neotyphodium gansuense from Drunken Horse Grass(Achnatherum inebrians)
Wang Lin1* Liao Fang2,3 Huang Guoming2 Liu Yueting2 Lou Jiafeng2 Zhou Qi1
1 Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing 101113, China ; 2 Tianjin Entry-Exit Inspection and Quarantine Bureau, Tianjin
300461, China ; 3 College of Life Sciences, Nankai University, Tianjin 300071, China
* Corresponding author, wanglin@bjciq.gov.cn
DOI: 10.3969/j.issn.1674-7968.2011.05.026
Abstract The infection of Neotyphodium gansuense in drunken horse grass (Achnatherum inebrians) has caused
great losses for livestock production in China. An effective molecular method for detection of N. gansuense is
quite necessary to set up to replace the present identification method based on morphology and isolation culture. In
this study, the N. gansuense isolated from the seed of drunken horse grass collected from Xinjiang, China was
confirmed by aniline blue staining microscopy and used as the environmental samples. The reference strain
collection used in this study included six species of Neotyphodium (N. gansuense, N. coenophialum, N. lolii, N.
huerfanum, N. chisosum and N. aotearoae), and all of them had highly similar morphological patterns. A fragment
of about 430 bp in size of the β-tubulin gene was first amplified and sequenced from each strain used. The
universal Taqman probe and its primers for genus Neotyphodium, and specific Taqman probe and its primers for N.
gansuense, were designed according to the sequence alignment results. The results showed the detection method
based on the double-colored Real-time fluorescence PCR developed in this study was an effective, rapid and
sensitive molecular method for detection of N. gansuense, which allowed us to detect the mycelia of N. gansuense
from a single grass seed, and the detection duration only needed about seven hours. This method can be used to
direct detect the infection rate of N. gansuense from the seeds of animal husbandry and lawn.
Keywords Molecular detection, Neotyphodium gansuense, β-tubulin gene, Double-colored Real-time
fluorescence PCR
双色荧光 PCR检测醉马草内生真菌(Neotyphodium gansuense)
汪琳 1* 廖芳 2, 3 黄国明 2 刘跃庭 2 罗加凤 2 周琦 1
1北京出入境检验检疫局，北京 101113；2天津出入境检验检疫局，天津 300461；3南开大学生命科学学院，天津 300071
*通讯作者，wanglin@bjciq.gov.cn
摘 要 醉马草内生真菌(Neotyphodium gansuense)曾对我国的畜牧业造成过巨大损失，目前其鉴定仍是基
于表型和分离培养的检测技术。本研究以与 N. gansuense形态非常相似的同属种 N. coenophialum、N. lolii、
N. huerfanum、N. chisosum和 N. aotearoae等共 6种 7个标准菌株，以及采自新疆的醉马草种子为环境材料，
扩增和测定了它们的 β-Tub基因约 430 bp的序列。根据序列分析结果，设计了 Neotyphodium属的通用
Taqman探针和引物以及 N. gansuense的特异探针和引物，基于双色荧光 PCR技术成功建立了快速、敏感的
分子检测方法。所建立的检测技术可实现对 N. gansuense菌丝培养物、单粒醉马草种子中 N. gansuense的检
测，并且检测时间只需 7 h。该检测技术能很好弥补传统检测鉴定方法的不足，可直接应用于国内外畜牧业种
子和草坪草种子中 N. gansuense带菌率的检测。
农业生物技术学报
Journal of Agricultural Biotechnology
The drunken horse grass, Achnatherum inebrians
(Hance) Keng, a perennial and numerous herb plant of
Gramineae, is an important potent poisonous weed
(Zhang and Li, 2006). This species is widely distributed
in natural grassland on the north-west China, although
it is reported to be originated in Europe and Asia areas.
This species has many characteristics, including strong
adaptability, cold- and drought-resistant. In recent
years, the grassland degradation caused by overgrazing
has resulted in the rapid spread of drunken horse grass.
It has become a major hazard to animal husbandry
because of its rapid spread in natural grassland (Deng et
al., 1998).
The drunken horse grass is toxic for horse, cattle,
sheep and other important livestock, especially
sensitive for horse (Zhang et al., 2006). In 2005,
drunken horse grass occurred in a large area in Alashan
County of Chinas Neimenggu has resulted in the
menace of more than 200 thousands livestock and the
death of at least 1000 livestock. The toxicity of drunken
horse grass is caused by alkaloids which is the
metabolity of the infected endophyte N. gansuense
(Bacon, 1988, 1995; Li and Zhang, 1998).
The endophyte N. gansuense in drunken horse
grass seed was first observed in the samples of Chinas
Xinjiang in 1994 by Bruehl et al, (1994). Miles et al,
(1996) successfully isolated the endophyte from the
drunken horse grass seed and seedling collected from
Chinas Xinjiang, then realized its pure culture.
Subsequently, many researchers focused on drunken
horse grass. Investigations performed by different
research groups showed that the infection rate of N.
gansuense reached 100% for drunken horse grass in
natural grassland of Chinas Xinjiang and Gansu (Li et
al., 1996; Li and Zhang, 1998; Li et al., 2004). The
isolated endophyte strain from the drunken horse grass
collected from Gansu of China was named as N.
gansuense Li et Nan (Li et al., 2004), while isolate from
Xinjiang of China was named as N. gansuense Li et
Nan var. inebrians, Moon & schardl (Moon et al.,
2007). The biological and physiological characteristics
of N. gansuense Li et Nan have been described by Li et
al.(2008).
In order to avoid poisoning of livestock, one of the
effective measures taken in production is to plant the
artificial grassland or replace the infected grassland by
using the seed of the carrier-free or low infection rate of
N. gansuense. As for the detection of the endophyte in
plant seed and seedling, the two present methods
including aniline blue staining microscopy (Li and
Zhang, 1998) and rose red staining microscopy (Saha et
al., 1988; Li et al., 1996) can not be accurately
identified to species level. Molecular methods
including general PCR and fluorescence PCR have
been successfully applied to detect pathogens including
N. coenophialum and N. lolii (Huang et al., 2007;
Prasad and Vidyarthi, 2009), providing an possible
effective molecular detection method for N. gansuense.
However, there has been no efficient detection method
for N. gansuense so far.
The double colored Real-time fluorescence PCR
is a new technology developed in recent years. Its more
sensitive, specificity and simplicity than conventional
PCR. The amplification and detection are closed in one
tube to avoid contamination. The duplex qualitative
PCR is widely used in the detection of animal and plant
diseases now(Huang et al, 2007). In order to effectively
manage the disease and prevent the pathogen from
spreading, this study is to establish an accurate, rapid,
sensitive and practical method based on the
double-colored Real-time fluorescence PCR for
detection of N. gansuense from drunken horse grass in
China.
1 Results and analysis
1.1 Isolation of the infected endophyte from
drunken horse grass seed
By aniline blue st aining microscopy as previously
described (Li and Zhang, 1998), the mycelia of the
infected endophyte showed an irregular distribution
pattern and rarely branched mycelium in the aleurone
layer cell gap of drunken horse grass seed. And a highly
similar colony morphology was shown on PDA culture
compared with that of reference strain
关键词 分子检测；醉马草内生真菌；β-Tub基因；双色荧光 PCR
974
双色荧光 PCR检测醉马草内生真菌(Neotyphodium gansuense)
Double-coloredReal-timeFluorescencePCRMethod forDetectionofNeotyphodium gansuense from Drunken Horse Grass
Figure 1 The amplification results of a part of Tub-2 gene
1~8: TJN1, TJN2, TJN3, TJN4, TJN5, TJN6, TJN7 and TJN9
respectively; 9: Negative control; M: DNA marker DL 2000
1 2 3 4 5 6 7 8 9 M
2000
1000
750
500
250
100
bp
Table 1 The sequences of the probes and primers used in this study
Probe
Tub-2-probe
49P-Probe
Primer
IS1
IS3
Tub-2-F
Tub-2-R
6F
94R
Sequence (5`~3`)
GGT GTT GAG CCC CCC TGA TTT
GTC TCA TCT AAG GGG CGG TAT
FAM-TTC TGG CAG ACC ATC TCT GGC GA-TAMRA
AGC TCG GAG GTA CCA TTG TA
CA AAC CGG TCA GTG CGT AA
JOE-TAC ACG CCC GCG GCC CAA-TAMRA
TGA GCC CCC CTG ATT TTG TAC
TCG GTC CGC CTC TCA TCA
(TCCMYA-1228), including white colony, extremely
slow growth, less aerial mycelium, rarely conidia.
Therefore, the isolated endophyte from Chinas
Xinjiang was identified as N. gansuense and used as
environmental samples to develop the molecular
detection method.
1.2 PCR amplification and sequence analysis of
Tub-2 gene
A fragment of about 430 bp in size of the Tub-2
gene was amplified from each of the eight strains used
in this study (Figure 1). The amplified fragments of
these strains except for strain TJN8 were selected to
sequence. Blastp analysis of the sequence determined
in this study showed 98% sequence identity with its
corresponding sequence, which suggested the sequence
determined in this study is from the Tub-2 gene. The
sequence of the Tub-2 gene of two N. gansuense strains
was identical to each other. The sequence of Tub-2
gene of N. gansuense was 431 bp including the primers,
within the range from 405 bp in N. huerfanum to 439
bp in N. chisosum.
The result of sequence alignment was shown in
Figure 2. Sequence comparison showed high sequence
similarity between them, from 92% (N. gansuense and
N. coe nophialum) to 98% (N. coenophialum, N.
chisosum and N. lolii). The variations among them
were mostly caused by the insertions or deletions,
which provide potential sites for developing the
molecular detection methods. According to the
sequence alignment result, the universal Taqman probe
and its primers for genus Neotyphodium, as well as
specific Taqman probe and its primers for N.
gansuense, were designed (Table 1; Figure 2).
1.3 Double-colored Real-time fluorescence PCR
detection of mycelia genomic DNA
Sequenc e analysis showed that the sequence of
theoretical amplicon for TJN1 and TJN2 by primer pair
6F/94R were identical to each other. So, seven strains
except for TJN2 were used to perform the
double-colored Real-time fluorescence PCR
amplification. The detection results of mycelial
genomic DNA using the double-colored Real-time
fluorescence PCR was shown in Figure 3. Data were
analyzed using the software SDS version 1.1 provided
by Applied Biosystems Inc, USA. When FAM
fluorescence marker was selected, the △Rn curves of
TJN1, TJN3, TJN4, TJN5, TJN6, TJN7 and TJN9
strains showed a positive growth whereas the △Rn
curve of negative control showed a straight line (Figure
3A), which suggested that the universal probe
Tub-2-probe designed in this study are effective for
detection of genus Neotyphodium including N.
gansuense, N. coenophialum, N. lolii, N. huerfanum, N.
chisosum and N. aotearoae. When JOE fluorescence
marker was selected, the △Rn curves of five similar
species (TJN3, TJN4, TJN5, TJN6 and TJN7) and
negative control showed a straight l ine whereas the
975
农业生物技术学报
Journal of Agricultural Biotechnology
△Rn curve of two N. gansuense strains (TJN1 and
TJN9) showed a positive growth (Figure 3B),
suggesting that the probe 49P are specific for detection
of N. gansuense strain.
1.4 Double-colored Real-time fluorescence PCR
detection of genomic DNA from a single grass seed
As shown in Figure 3C and 3D, the results of
double-colored Real-time fluorescence PCR detection
for genomic DNA from a single grass seed were
identical to those for genomic DNA from mycelia. The
only difference between them was that the △Rn curve
for a single grass seed was lower than that for mycelia.
The results showed that the probe Tub-2-probe and
probe 49P were universal for genus Neotyphodium and
specific for N. gansuense, respectively, which were
effective for detection of a single grass seed of N.
gansuense by double-colored Real-time fluorescence
PCR.
1.5 Repeatability of detection method established
Among 90 seeds used, only one seed infected with
endophyte by aniline blue staining microscopy
detection showed an unsatisfactory result by the
double-colored Real-time fluorescence PCR detection.
Fluorescence PCR detection of uninfected seed showed
a negative result(Data not shown). We guessed that the
extraction of genomic DNA failed. These results
suggested that the established double-colored Real-time
fluorescence PCR detection method for N. gansuense in
drunken horse grass had a good reproducibility.
2 Discussions
As an important potent poisonous weed (Zhang
and Li, 2 006), the drunken horse grass has become a
major hazard to animal husbandry (Deng et al., 1998).
The present identification method for detection of N.
gansuense was based on morphology and isolation
culture (Li and Zhang, 1998; Li et al., 2008), which
would not only need the complicated procedures but
also spend more than one month. Moreover, N.
gansuense produced less aerial mycelium and rarely
conidia (Li et al., 2003), which made very difficulty for
its identification. Many recent research results (Huang
et al, 2007) showed that detection method of plant
pathogens based on the rapid, specific and sensitive
Real-time fluorescence PCR could meet well to the
shortfalls of traditional detection method based on
morphology and isolation culture. The parasitic N.
gansuense is located at the epidermis of seed, so the
genomic DNA of the infected endophyte can be
extracted together with that of the host.
In this study, we first amplified and sequenced the
part of Tub-2 gene for seven reference strains and one
isolated strain. The results of double-colored Real-time
fluorescence PCR detection showed that an effective,
rapid and sensitive molecular detection method of N.
gansuense was successfully developed. This method
allows us to detect the mycelia of N. gansuense
cultured, also to direct detect the grass seeds which are
infected with the endophyte. A single grass seed
infected with the endophyte can be detected by the
method established in this study. The detection method
based on the double-colored Real-time fluorescence
PCR can shorten the detection duration at least one
month of traditional method to about seven hours,
which can improve the detection efficiency in practical.
This method can be used to direct detect the infection
Code
TJN8
TJN1
TJN2
TJN3
TJN4
TJN5
TJN6
TJN7
Species
Neophodium gansuense
N. gansuense Li et Nan var. inebrians
N. gansuense Li et Nan
N. coenophialum
N. lolii
N. huerfanum
N. chisosum
N. aotearoae
Host
Achnatherum inebians
A. inebians
A. inebians
Festuca arundinacea
Lolium perenne
Festuca arizonica
Stipa eminens
Echinopogon ovatus
Origin
This study
ATCCMYA-1228
ATCCMYA-3669
ATCC56422
ATCCMYA-3408
ATCC64040
ATCC64037
ATCCMYA-1229
GenBank accession No.
GQ422359
GQ422360
GQ422361
GQ422365
GQ422363
GQ422362
GQ422364
Table 2 Tested strains in the study
976
6F 49P-probe
N.gansuense(TJN1) GGTGTTGAGCCCCCCTGATTTTGTACCCCGCCGGGCCCGGCCACGACGAC G CG C AATG A AATC TG T GAGGC
N.gansuense(TJN2) GGTGTTGAGCCCCCCTGATTTTGTACCCCGCCGGGCCCGGCCACGACGAC G CG C AATG A AATC TG T GAGGC
N.coenophialum GGTGTTGAGCCCCCCTGATTTCGTACCCCGCCGAGCCCGGCCACGACGTGCACGCCC-------AACGAACAGTCGTGATGAGAGGC
N.lolii GGTGTTGAGCCCCCCTGATTTCGTACCCCGCCGAGCCCGGCCACGAAGTGCACGCCC-------AACGAACAGTCGTGATGAGAGGC
N.chisosum GGTGTTGAGCCCCCCTGATTTCGTACCCCGCCGAGCCCGGCCACGACGTGCACGCCC-------AACGAACAGTCGTGATGAGAGGC
N.aotearoae GGTGTTGAGCCCCCCTGATTTCGTACCCCGCCGAGCCCGACCACGACGTGCACGCCC-------AACGGACAGTCGTGATGAGAGGC
N.huerfanum GGTGTTGAGCCCCCCTGATTTCGTACCCCGCCGAGCCCGGCCACGACGTGCACGCCC-------AACGGACAGTCGTGATGAGAGGC
********************* *********** ***** ****** ** ******* ** * *** **************
96R
N.gansuense(TJN1) GGACCGAGACAGAATTAATGATTGCGGTATTTCGAGAACTGTAGCTGATCCATTT---------TCTTTCCCTCTAGGTTCATCTTC
N.gansuense(TJN2) GGACCGAGACAGAATTAATGATTGCGGTATTTCGAGAACTGTAGCTGATCCATTT---------TCTTTCCCTCTAGGTTCATCTTC
N.coenophialum GGATCGAGACAAAATTAATGAATGCGGTATT-CGAGAACTGTAGCTGACCTGTTTCTTCCCCTCTTTTCCCCTCTAGGTTCATCTTC
N.lolii GGACCGAGACAAAATTAATGAATGCGGTATT-CGAGAACTGTAGCTGACCTGTTTCTTTCCCTCTTTTCCCCTCTAGGTTCATCTTC
N.chisosum GGACCGAGACAAAATTAACGAATGCGGTATT-CGAGAACTGTAGCTGACCTGTTTCTTTCCCTCTTTTCCCCTCTAGGTTCATCTTC
N.aotearoae GGACCGAGACAAAATGAATGAATGCGGTATT-CGAGAACTGTAGCTGACCTTTTT---------TCTTTCCCTCTAGGTTCATCTTC
N.huerfanum GGACCGAGACAACATCATTGAATGCGGTATT-CGAGAGCTGTAGCTGACCTTTTT----------CTTTCCCTCTAGGTTCATCTTC
*** ******* ** * ** ********* ***** ********** * *** ** ******************
Tub-2-R
N.gansuense(TJN1) AGACCGGTCAGTGCGTAAGTGACAAATTCGCCGACCTCGAACGACAG----GCAGAAACAACATGAAAA--CTCACATTTATTTGGG
N.gansuense(TJN2) AGACCGGTCAGTGCGTAAGTGACAAATTCGCCGACCTCGAACGACAG----GCAGAAACAACATGAAAA--CTCACATTTATTTGGG
N.coenophialum AAACCGGTCAGTGCGTAAGTGACAAAT-CGCCGACCTCGAACGACAG----GCACAAACAGCATGAAAAAACTCACATTCATTTGGG
N.lolii AAACCGGTCAGTGCGTAAGTGACAAATTCGCCGACCTCGAACGACAG----GCACAAACAGCATGAAAAA-CTCACATTCATTTGGG
N.chisosum AAACCGGTCAGTGCGTAAGTGACAAATCCGCCGACCTCGAACGACAGACAGGCACAAACAGCATGAAAAA-CTCACATTCATTTGGG
N.aotearoae AAACCGGTCAGTGCGTAAGTGACAAATCCGCCGACCTCGAACGACAG----ACACAAATAACATGAAAAA-CTCACATTTATTTGGG
N.huerfanum AAACCGGTCAGTGCGTAAGTGACAAATCTGCCGACCTCGAACGACAG----GCACAAATAACATGAAAAA-CTCACATTGATTTGGG
* ************************* ****************** ** *** * ******** ******** *******
Tub-2-probe Tub-2-F
N.gansuense(TJN1) CAGGGTAACCAAATTGGTGCTGCTTTCTGGCAGACCATCTCTGGCGAGCACGGCCTCGACAGCAATGGTGTGTACAATGGTACCTCC
N.gansuense(TJN2) CAGGGTAACCAAATTGGTGCTGCTTTCTGGCAGACCATCTCTGGCGAGCACGGCCTCGACAGCAATGGTGTGTACAATGGTACCTCC
N.coenophialum CAGGGTAACCAAATTGGTGCTGCTTTCTGGCAGACCATCTCTGGCGAGCACGGCCTCGACAGCAATGGTGTGTACAATGGTACCTCC
N.lolii CAGGGTAACCAAATTGGTGCTGCTTTCTGGCAGACCATCTCTGGCGAGCACGGCCTCGACAGCAATGGTGTGTACAATGGTACCTCC
N.chisosum CAGGGTAACCAAATTGGTGCTGCTTTCTGGCAGACCATCTCTGGCGAGCACGGCCTCGACAGCAATGGTGTGTACAATGGTACCTCC
N.aotearoae CAGGGTAACCAAATTGGTGCTGCTTTCTGGCAGACCATCTCTGGCGAGCACGGCCTCGACAGCAATGGTGTGTACAATGGTACCTCC
N.huerfanum CAGGGTAACCAAATTGGTGCTGCTTTCTGGCAGACCATCTCTGGCGAGCACGGCCTCGACAGCAATGGTGTGTACAATGGTACCTCC
***************************************************************************************
N.gansuense(TJN1) GAGCTCCAGCTCGAGCGTATGAGTGTCTACTTCAACGAGGCAAGTTTTCATAATCAAAAAAGTCTCCATTGAGCTGC----ATACCG
N.gansuense(TJN2) GAGCTCCAGCTCGAGCGTATGAGTGTCTACTTCAACGAGGCAAGTTTTCATAATCAAAAAAGTCTCCATTGAGCTGC----ATACCG
N.coenophialum GAGCTCCAGCTCGAGCGTATGAGTGTCTACTTCAACGAGGTAAGTCTTCATAATCTAAA--GTCTCCGTTGAGCTACATACATACCG
N.lolii GAGCTCCAGCTCGAGCGTATGAGTGTCTACTTCAACGAGGTAAGTCTTCATAATCTAAA--GTCTCCATTGAGCTACATACATACCG
N.chisosum GAGCTCCAGCTTGAGCGTATGAGTGTCTACTTCAACGAGGTAAGTCTTCATAATCTAAA--GTCTCCATTGAGCTACATACATACCG
N.aotearoae GAGCTCCAGCTCGAGCGTATGAGTGTCTACTTCAACGAGGTAAGTCTTCATAATCTAAA--GTCTCCATTGAGCTAC----ATACCG
N.huerfanum GAGCTCCAGCTCGAGCGTATGAGTGTCTACTTCAACGAGGCAAGTCT------------------CCATTGAGCTAC----ATACCG
*********** ******************** ******** **** * ** ******* * ******
N.gansuense(TJN1) CCCCGGAGATGAGAC
N.gansuense(TJN2) CCCCGGAGATGAGAC
N.coenophialum CCCCGGAGATGAGAC
N.lolii CCCCGGAGATGAGAC
N.chisosum CCCCGGAGATGAGAC
N.aotearoae CCCCGGAGATGAGAC
N.huerfanum CCCCGGAGATGAGAC
***************
Figure 2 The result of sequence alignment of partial Tub-2 gene
The universal Taqman probe was highlighted with viridian and its amplification primers was highlighted with dark green for genus
Neotyphodium; Specific probe was underlined and red-clolored and its primers was underlined for N. gansuense
双色荧光 PCR检测醉马草内生真菌(Neotyphodium gansuense)
Double-coloredReal-timeFluorescencePCRMethod forDetectionofNeotyphodium gansuense from Drunken Horse Grass 977
农业生物技术学报
Journal of Agricultural Biotechnology
rate of N. gansuense from the seeds of animal
husbandry and lawn.
3 Materials and methods
3.1 Strain collection
The se eds of drunken horse grass (Achnatherum
inebrians) used in this study were collected from
Xinjiang, China. The Neotyphodium gansuense (TJN8)
was isolated and confirmed by microscopy after aniline
blue staining according to the method previously
described (Mile et al., 1996). The reference strains of
N. gansuense (TJN 1, 2) and five Neotyphodium species
(N. coenophialum, N. lolii, N. huerfanum, N. chisosum
and N. aotearoae), which have highly similar
morphological patterns, and all of them were used as
the reference strain collection (Table 2). All these
reference strains were purchased from American Type
Culture Collection (ATCC).
3.2 DNA extraction
All the strains including the 7 reference strains and
1 isolated strain (TJN8) used in this study were
incubated as previously described (Liu et al., 2005).
The mycelia were collected by centrifuge and used to
extract DNA using Universal Genomic DNA
Mini-Isolation Kit (Shanghai Sangon Biological
Engineering Technology & Services Co., Ltd., China).
This Kit was also used to extract genomic DNA from a
single grass seed. The DNA templates were diluted to
be a final concentration of 50 ng/μL by measuring on
BIO-RAD nucleic acid and protein analyzer (Bio-Rad
Laboratories, Inc., USA).
3.3 Preparation of probes and primers
A fragment of about 430 bp in size of the beta
tubulin (Tub-2) gene was first amplified and sequenced
for each of the eight strains according to the previously
reported (Doss et al., 1998). PCRs were performed in a
30 μL volume with 0.5 μL of rTaq (TaKaRa
Biotechnology Co., Ltd., Dalian, China), 1 μL of
template DNA, 2.5 μL of 10×PCR buffer with MgCl2, 1
μL of dNTPs (2.5 mmol/L for each), 1 μL of each
primer (20 pmol). The ddH2O was added to replace the
template DNA as the negative control. The PCR
Figure 3 Double-colored Real-time fluorescence PCR detection results
A, B: Mycelial genomic DNA with the universal probe for the genus Neotyphodium and the specific probe for N. gansuense,
respectively; C, D: Genomic DNA for a single grass seed with the universal probe for the genus Neotyphodium and the specific probe
for N. gansuense, respectively
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amplification was performed on Biometra Thermal
Cycler (BioPortfolio Ltd, Germany) under the
following procedure: 1 min at 94℃ , followed by 35
cycles of 15 sec at 94℃ , 60 sec at 60℃ , with a
subsequent 10 min final extension at 72℃ . PCR
products were detected by 1% agarose gels. The PCR
fragments were directly sequenced in both directions.
DNA sequencing was performed on an ABI 3730
Genetic Analyzer (Applied Biosystems Inc, USA) by
Invitrogen Corporation (Shanghai, China). The
sequence data have been deposited in GenBank under
accession no. GQ422359 - GQ422365.
The eight sequences of Tub-2 gene determined in
this study were aligned by using Clustal X version 1.8
(Thompson et al., 1997). The Primer Express software
version 2.0 was used to design the probes and primers.
For genus Neotyphodium, the universal probe,
Tub-2-probe, and one pair of primers, Tub-2-F and
Tub-2-R, were produced based on a highly conserved
sequence alignment region. For species N. gansuense,
the specific probe, 49P-probe, and one pair of primers,
6F and 94R, were produced based on a highly variable
sequence alignment region. The sequences of the
probes and primers used in this study were listed in
Table 2. All the probes and primers were synthesized
by Invitrogen Corporation (Shanghai, China).
3.4 Development of the double-colored Real-time
fluorescence PCR detection method
The Real-time fluorescence PCR Kit (ExTaqTM
R-PCR version 2.1) (TaKaRa Biotechnology Co., Ltd,
Dalian, China) and ABI 7000 Real-time fluorescence
PCR Systems (Applied Biosystems Inc, USA) were
used for fluorescence PCR amplifications. Each
reaction in a volume of 25 μL contained 0.25 μL of
template DNA, 0.5 μL of ExTaq, 2.5 μL of 10×ExTaq
buffer with MgCl2, 1 μL of dNTPs (2.5 mmol/L for
each), 1 μL of each primer (10 pmol; Tub-2-F/Tub-2-R
and 6F/94R), 1 μL of probe (20 pmol; Tub-2-probe and
49P). The ddH2O was added to replace the template
DNA as the negative control. The amplification
program was performed with the following conditions:
10 min at 95℃; 45 cycles of 15 s at 95℃, 60 s at 60℃.
Put all the DNA samples on the reaction plate of
real-time fluorescence PCR machine, set reaction
conditions, let the PCR machine automatically monitor
all the process of PCR reaction in real time and record
the data. When the reaction completed, open the
analysis software and set a baseline and a threshold
value.
For genomic DNA from a single grass seed, the
double-colored Real-time fluorescence PCR
amplification volume and program were identical to
that abovementioned for mycelia genomic DNA, with
the only difference in the volume of template DNA (1.5
μL of genomic DNA from a single grass seed).
The repeatability experiment was performed
independently three times. Thirty seeds of drunken
horse grass were selected randomly for each
experiment. Each seed was detected by microscopy
after aniline blue staining. Total DNA from a single
grass seed including plant seed and possible infected
endophyte was extracted as the template DNA for the
double-colored Real-time fluorescence PCR
amplification.
References
Bacon C.W., 1988, Procedure for isolating the endophyte from
tall fescue and screening isolates for ergot alkaloids, Applied
and Environmental Microbiology, 54(11): 2615~2618
Bacon C.W., 1995, Toxic endophyte-infected tall fescue and
renge grasses: historic perspectives, Journal of Animal Sci-
ence. 73(3): 861~870
Bruehl G.W., Kaiser W.J., and Klein R.E., 1994, An endophyte
of Achnatherum inebrians, an intoxicating grass of north-
west China, Mycologia, 86(6): 773~776
Deng K.D., Peng H.H., Li W.R., Warren B.E., and Fletcher I.C.,
1998, The ergonovine content and the nutritive value of am-
moniated drunken horse grass with urea, Pratacultural Sci-
ence (Chinese),15(4): 10~13
Doss R.P., Clement S.L., Kuy S.R., and Welty R.E., 1998, A
PCR-based technique for detection of Neotyphodium endo-
phyte in diverse accessions of tall fescue, Plant Disease. 82
(7): 738~740.
Huang G.M., Liao F., Liu Y.T., Cui T.J., and Luo J.F., 2007, De-
tection of Neotyphodium coenophialum and N. lolii based on
real-time fluorescence PCR, Mycosystema (Chinese ), 26:
257~265
Li B.J., Zheng X.H., and Sun S.C., 1996, An investigation of en-
dophte-grasses in north-west of China, Grassland of China
双色荧光 PCR检测醉马草内生真菌(Neotyphodium gansuense)
Double-coloredReal-timeFluorescencePCRMethod forDetectionofNeotyphodium gansuense from Drunken Horse Grass 979
农业生物技术学报
Journal of Agricultural Biotechnology
(Chinese), 27(2): 29~32
Li C.J., Gao J.H., and Ma B., 2003, Seven diseases of drunken
horse grass (Achnatherum inebrians) in China, Pratacultural
Science (Chinese), 20(11): 51~53
Li C.J., Nan Z.B., and Li F., 2008, Biological and physiological
characteristics of Neophodium gansuense symbiotic with
Achnatherum inebrians, Microbiological Research, 163 (4):
431~440
Li C.J., Nan Z.B., Liu Y., volk H.P., and Dapprich P., 2008,
Methodology of endophyte detection of drunken horse grass
(Achnatherum inebrians), In Proceedings of the Annual
Meeting of Chinese Society for Plant Pathology, Beijing
China Agricultural Science Technology Press, Beijing, pp.
105~108
Li C.J., Nan Z.B., paul V.H., Peter D.,and Liu Y., 2004, A new
Neotyphodium species symbiotic with drunken horse grass
(Achnatherum inebrians) in China, Mycotaxon, 90 (1):
141~147
Li X.S., and Zhang X.Z., 1998, The relationship between toxic
components of drunken horse grass and its endophyte,
Grass-Feeding Livestock (Chinese), 101(4): 44~46
Liu Y.T., Cui T.J., Huang G.M., and Liao F.,2005, Study on en-
dophytic fungi in imported Festuca arundiacea and Lolium
perenne, Plant Quarantine (China), 19(4): 193~197
Miles C.O., Lane G.A., di Menna M.E., Garthwaite I., Piper E.L.,
Ball O.J.P., Latch G.C.M., Allen J.M., Hunt M.B., Bush L.
P., Min F.K., Fletcher I., and Harris P.S.,1996, High levels
of ergonovine and lysergie acid amide in toxic Achnatherum
inebrians accompany infection by an Acremonium-like en-
dophytic fungus, Journal of Agricultural Food Chemistry, 44
(5): 1285~1290
Moon C.D., Guillaumin J.J., Ravel C., Li C.J., Craven K.D., and
Schardl C.L., 2007, New Neotyphodium endophyte species
from the grass tribes Stipeae and Melieeae, Mycologia, 99
(6): 895~905
Prasad D., and Vidyarthi A.S., 2009, DNA based methods used
for characterization and detection of food borne bacterial
pathogens with special consideration to recent rapid meth-
ods DNA based methods used for characterization and de-
tection of food borne bacterial pathogens with special con-
sideration to recent rapid methods, African Journal of
Biotechnology,8 (9): 1768~1775
Saha D.C., Jackon M.A., and Johnson-Cicalese J.M., 1988, A
rapid staining method for detection of endophyte fungus in
turf grasses, Phytopathology, 78: 237~239
Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., and
Higgins D.G., 1997, The CLUSTAL_X windows interface:
flexible strategies for multiple sequence alignment aided by
quality analysis tools, Nucleic Acids Research, 25 (24):
4876~4882
Zhang W., and Li G., 2006, Toxic components and sections
screened out from fragments extracted from Achnatherum ine－
brians, Progress in VeterinaryMedicine (China), 27(7): 97~99
Zhang W., Li G., and Li X.F., 2006, Study on extract of toxic
components from Achnatherum inebrians (Hance) Keng,
Biotechnology (Chinese),16(6): 60~62
980