Advance Journal of Food Science and Technology 4(6): 426-429, 2012
ISSN: 2042-4868; E-ISSN: 2042-4876
© Maxwell Scientific Organization, 2012
Submitted: September 13, 2012
Accepted: October 09, 2012
Published: December 20, 2012
Investigation of Terahertz Spectral Signatures of DNA of Pine Wood Nematode
1
Ling Jiang, 1Chun Li, 2Lin Huang, 3Zhenwei Zhang, 3Cunlin Zhang and 1Yunfei Liu
1
Department of Information Science and Technology,
2
Department Forest Resources and Environment, Nanjing Forestry University, Nanjing 210037, China
3
Terahertz Laboratory, Department of Physics, Capital Normal University, Beijing, China
Abstract: In this study, we present the fingerprint characteristic about the identification of harmful Bursaphelenchus
xylophilus (Bx) nematode and harmless Bursaphelenchus mucronatus (Bm) nematode by applying terahertz
spectroscopic techniques. We measure the transmission of the Deoxyribose Nucleic Acid (DNA) of the Bx and the
Bm samples and their corresponding Polymerase Chain Reaction (PCR) amplification segments of the DNA
molecules by the Terahertz Domain Spectroscopy (THz-TDS) and the Fourier Transform Infrared spectroscopy
(FTIR). The low-frequency absorption features measured by THz-TDS exhibits different absorption peaks (i.e., 0.07
and 0.11 THz) for the Bx and Bm samples respectively. The calculated relative refractive index of the Bm-PCR
segment is found to be much smaller compared with other three samples. The higher frequency characteristic of the
four samples measured by the FTIR spectroscopy shows similar absorption peaks, except that the Bm-DNA provides
a smooth differential feature of absorption at 5.46 THz. These measurement results indicate the Bm samples
including DNA and PCR segment indeed have unique signature behaviors different from the Bx samples. It
demonstrates that the terahertz spectroscopic technique is a useful method to distinguish the Bx and the Bm’s DNA
and PCR segments by locating the absorption frequency.
Keywords: Absorption frequency, Bm nematode, Bx nematode, terahertz spectroscopy
spectroscopic method to study the absorption
characteristics of the DNA and its PCR amplification
segment of the Bx and Bm nematodes, i.e., Bx-DNA,
Bx-PCR, Bm-DNA and Bm-PCR samples by using
Terahertz Time Domain Spectroscopy (THz-TDS) and
Fourier Transform Infrared Spectroscopy (FTIR). This
is a continued study based on our previous study (Liu
et al., 2009a, b, 2010). In this study, we obtain the
different characteristic peaks of the Bm and the Bx
samples. The experimental results will be analyzed by
phonon modes in DNA macromolecules and dielectric
resonances in terahertz spectra.
INTRODUCTION
The pine wood nematode, Bursaphelenchus
xylophilus (Bx), is called pine cancer, which is a
serious pine wood disease. Bursaphelenchus
mucronatus (Bm) is a harmless insect living in the
healthy pine wood, which is quite similar to the Bx in
shape. In order to diagnose the Bx nematode disease in
early stage, it’s necessary to distinguish the two
nematodes. Currently the conventional method is realtime fluorescence Polymerase Chain Reaction (PCR)
technique, which focus on the investigation of the PCR
segment of Deoxyribose Nucleic Acid (DNA) molecule
of the Bx and the Bm samples by introducing the
fluorescence marker (Cao et al., 2005; Wang et al.,
2006). This method has several defects, for instance,
time-consuming, high cost, relative high error rate due
to the fluorescence marker. It’s mainly employed for
quarantine purposes. To early diagnose the pine wilt
disease, it’s necessary to find a fast and exact method.
Terahertz spectroscopic technology has been
successfully used to biology and medicine to study the
animal and plant protein, nucleic acid and amino acid
and soon Markelz et al. (2000). However, the spectral
property of DNA has not been investigated thoroughly
due to its huge mass. In this study, we employ the THz
MEASUREMENT SETUP AND METHODS
Measurement setup: The characterization of the BxDNA, Bx-PCR, Bm-DNA and Bm-PCR samples are
performed by the THz-TDS and the FTIR spectroscopy
respectively. By measuring the change of the intensity
of probe beam, we can obtain the THz wave signal
which carries the information of the sample in the
frequency range of 0-2.5 THz. In order to understand
the spectral property of DNA molecule at high
frequencies, we measure its absorbance by using
Fourier Transform Infrared spectroscopy (FTIR), which
is a technique used to obtain an infrared spectrum of
Corresponding Author: Ling Jiang, Department of Information Science and Technology, Nanjing Forestry University, Nanjing
210037, China
426
Adv. J. Food Sci. Technol., 4(6): 426-429, 2012
absorption, emission, photoconductivity or Raman
scattering of a solid, liquid or gas. The conventional
FTIR system consists of optical source, Michelson
Interferometer and detector. In our measurement, the
transmission spectral data were obtained. The
measurement setup and principle of the THz-TDS and
FTIR systems were introduced in previous study (Liu
et al., 2010).
Sample preparation: The Bx-DNA, Bm-DNA, BxPCR and Bm-PCR samples were measured by the THzTDS and FTIR spectroscopy respectively. The Bx-DNA
and the Bm-DNA were distilled from the Bx and the Bm
nematodes by biological methods. The Polymerase
Chain Reaction (PCR) is a technique in molecular
biology to amplify a single or few copies of a piece of
DNA across several orders of magnitude, generating
thousands to millions of copies of a particular DNA
sequence. The Bx-PCR and the Bm-PCR samples are
the special segments extracted from the Bx- and BmDNA molecules. The pure Bx-DNA, Bm-DNA, Bx-PCR
and Bm-PCR samples have the weight about 20 μg,
which were mixed with the Polyethylene (PET) powder
and compressed into a thin pill under high pressure. The
final pill has a 0.5-mm thickness, 13-mm diameter, 30mg weight, after drying by vacuum drying oven until
the water content is below 5%. The pure PET powder is
also made into a pill with same thickness and weight as
the other four samples for comparison. Given the PET
is transparent to THz signal; the measured results will
reflect the absorption characteristics of the Bx and the
Bm nematodes.
Data analysis methods: In the THz-TDS
measurement, the refractive index ns (ω), absorption
coefficient αs (ω) and extinction coefficient ks (ω) of the
four samples are obtained by means of the physical
model developed by the research groups (Duvillaret
et al., 1999). Assuming that ETHz (ω) is the incident
field, Eref (ω) the reference field and Esam (ω) the field
where the signal passes through the sample. The
reference field can be given by:
~
Eref ( )  ETHz ( )e  jn ( )L / c
where, n~s and n~0 is the complex refractive indices of
the sample and the medium around the sample
respectively,
with
the
expression
of
n~  n ( )  j ( ).
For thick samples with the thickness of the order of
millimeter, the Fabry-Perot effect can be ignored.
Therefore, the field Esam (ω) passing through the sample
is:
~
Esam ( )  ETHz ( )tos e  jns ( )d / c t so
where, d is the thickness of the sample.
In our experiment, refractive index
H ( )  Esam ( ) / Eref ( )
~
 4n~ ( )e  jd ( ns 1) / c /(1  n~ ) 2
s
(2)
is
(4)
s
  ( )e  j ( )
where ρ(ω) and (ω) are the ratio of the amplitude and
the phase difference between the sample and the
reference which are obtained from the THz-TDS
experiment. Considering κb<<nb, we obtain:
 ()  4ns ()e ( ) d / c /(ns ()  1) 2
(5)
 ( )  ( ns ( )  1)d / c
(6)
s
Finally, the expressions of the refractive index
ns(ω), the absorption coefficient αs(ω) and the
extinction coefficient ks(ω) can be achieved as follows:
n s ( )   ( ) 
 s ( ) 
c
d
1
4n s ( )
2
)
 ln(
d
 ( )  (n s ( )  1) 2
k s ( ) 
where,
L : The distance where the reference wave propagates
in linear (uniform) medium
c : The velocity of light
t os ( )  2 n~s ( n~0  n~s )
n~0
approximately 1. Substituting Eq. (2) into (3), the ratio
of Eref (ω) to Esam (ω) is given by:
(1)
When the THz wave incidence on the sample
vertically, based on the Fresnel’s equations the
functions of transmission coefficient tos(ω) can be
written as:
(3)
s c c
4n s ( )

 ln(
)
2
d
 ( )  (n s ( )  1) 2
(7)
(8)
(9)
MEASUREMENT RESULTS
THz transmission measured by THz-TDS: We have
employed the conventional THz-TDS spectroscopy to
measure the transmission characteristics of the BxDNA, Bm-DNA, Bx-PCR and Bm-PCR samples. Figure
1 display the measured time domain spectra of the BxDNA and the Bm-DNA mixed with the PET powder.
The time domain spectra of the Bx-PCR and Bm-PCR
427 Adv. J. Food Sci. Technol., 4(6): 426-429, 2012
Fig. 1: Time domain spectra of the Bx-DNA and Bm-DNA
mixed with the PET powder
Fig. 2: Fourier transform spectra of the Bx-DNA, Bx-PCR,
Bm-DNA, Bm-PCR samples after the correction of the
pure PET powder in the frequency range of 0-2.5 THz
Fig. 3: Relative refractive index as a function of frequency for
the Bx-DNA, Bx-PCR, Bm-DNA, Bm-PCR samples,
based on the refractive index of the pure PET sample
were measured to be similar to that of DNA samples,
all of which have time delay compared with the case of
Fig. 4: Absorption as a function of frequency for the BxDNA, Bx-PCR, Bm-DNA, Bm-PCR samples by using
FTIR
the pure PE powder. The calculated FTS responses of
the four samples are shown in Fig. 2 after the correction
of the pure PET powder. The four samples exhibit
different absorption peaks, with the Bx-PCR and BxDNA at 0.07 THz and the Bm-PCR and Bm-DNA at
0.11 THz. The discrepancy of the absorption peak may
originate from the dielectric resonances of phonon
modes of DNA and its segments.
According to the Eq. (7) and (8), the refractive
index and absorption coefficient were calculated for the
Bx-DNA, Bx-PCR, Bm-DNA, Bm-PCR samples, as
shown in Fig. 3. It should be noted that the calculated
refractive index are relative ones based on that of the
pure PET sample. The calculated refractive index of the
Bm-PCR sample is quite different from the other three
samples with the discrepancy of lager than 0.1.
The measurement by THz-TDS indicate the Bm
and the Bx DNA samples have absorption features at
very lowest terahertz frequency regions. Generally the
elastic nature of DNA polymer results in structural
vibrations that are acoustic modes below approximately
0.01 THz. DNA phonon modes in the microwave and
millimeter-wave regime, with frequency smaller than
0.1 THz, are for the most part optically inactive and
absorption spectrum from this region cannot be
expected to yield a great deal of information about the
internal structure of the polymer (Woolard et al., 2002).
However, the absorption peaks at 0.07 and 0.11 THz
are observed for the Bm and the Bx samples
respectively. It means at the lower edge of millimeterwave DNA phonon modes reflect the structural
information. As a supplement of the whole terahertz
region, we make the measurement at higher frequencies
above 3 THz by FTIR spectroscopy.
Transmission measured by FTIR: We measure the
response above 1 THz by using broadband FTIR
spectroscopy. In the frequency range of 1-10 THz, the
measured FTS responses exhibit several similar peaks
for the four samples. Figure 4 displays the absorbance
428 Adv. J. Food Sci. Technol., 4(6): 426-429, 2012
THz derived from the absorption characteristic. This
investigation will provide a new method based on
terahertz spectroscopy technology to detect pine wood
nematode.
ACKNOWLEDGMENT
Fig. 5: Differential plot of the absorbance characteristic for
the Bm-PCR, Bm-DNA, Bx-PCR, Bx-DNA
of the four samples in terms of FTIR spectroscopy. In
order to find the signature feature to discriminate the
Bm and the Bx, the differential plots of the transmission
characteristic versus frequency (i.e., dT/df calculated
from smoothing over nine data points) from the DNA
and PCR segments of the Bm and Bx are compared, as
shown in Fig. 5. Here, approximately similar results for
the four different samples are observed. Furthermore,
the differential plots reveal a discrepancy at 5.46 THz
between Bm-DNA and the other three samples. The
Bm-DNA shows a smooth curve while the other three
samples exhibit a weak shoulder feature, which
probably results from soft phonon modes that are
obscured by the absorption feature at Fig. 4.
According to the above measurement by TDS and
FTIR, the absorption frequency peaks at 0.07, 0.11 and
5.46 THz provide the signature features to distinguish
the Bm and the Bx DNA and PCR sample.
CONCLUSION
We have measured the time domain and frequency
domain responses of the Bx-DNA, Bx-PCR, Bm-DNA
and Bm-PCR by using the Terahertz Time Domain
Spectroscopy (THz-TDS) and the Fourier Transform
Infrared spectroscopy (FTIR). The Bx-PCR and BxDNA have an absorption peak at 0.07 THz, however
the Bm-PCR and Bm-DNA at 0.11 THz, suggesting that
the low-frequency collective modes indeed are terahertz
active. The calculated relative refractive index of the
Bm-PCR is smaller than that of another three samples.
Further measurement of the absorption characteristics
and differential curves at higher terahertz frequencies
above 1 THz by the FTIR demonstrates a different
feature exist of the Bm-DNA at 5.46 THz, which
indicate different phonon modes existing in the BmDNA. Therefore, we can clearly distinguish the Bm and
the Bx nematodes by observing the low frequency peak
at 0.07 and 0.11 THz and the differential peak at 5.46
This study is supported by the National Natural
Science Foundation of China under Contracts 31170668
and 31200541, by the Natural Science Foundation of
Jiangsu province under contract BK2012417, provincial
university foundation of Jiangsu under Contract
10KJB510006, by the returned personnel foundation of
ministry of education and by the fund of high level and
returned personnel of Nanjing Forestry University.
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