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RH: SHORT COMMUNICATIONS
A Loop-Mediated Isothermal Amplification (LAMP) Assay Targeting 16S rRNA
Gene for Rapid Detection of Anaplasma phagocytophilum Infection in Sheep and
Goats
Jinhong Wang, Yan Zhang, Xiaoxing Wang, Yanyan Cui, Yaqun Yan, Rongjun
Wang, Fuchun Jian, Longxian Zhang, and Changshen Ning
College of Animal Science and Veterinary Medicine, Henan Agricultural University,
Zhengzhou, 450002, China. Correspondence should be sent to Prof. Changshen Ning
at: [email protected]
Abstract: Anaplasma phagocytophilum is a zoonotic pathogen and the causative agent
of human granulocytic anaplasmosis (HGA) in humans and tick-borne fever in
various kinds of animals. In the present study, a loop-mediated isothermal
amplification (LAMP) assay for rapid detection of A. phagocytophilum was
developed using primers specific to 16S rRNA gene of this organism. The LAMP
assay was performed at 65 C for 60 min and terminated at 80 C for 10 min. The
optimal reaction conditions, under which no cross-reaction was observed with other
closely related tick borne parasites (Anaplasma bovis, Anaplasma ovis, Theileria
luwenshuni, Babesia motasi and Schistosoma japonicum) was established. The assay
exhibited much higher sensitivity when compared with conventional PCR (1 copy vs.
1,000 copies). To evaluate the applicability of the LAMP assay, 94 sheep field blood
samples were analyzed for A. phagocytophilum infection using LAMP, nested PCR
and conventional PCR assay at the same time. A positive LAMP result was obtained
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from 53 of the 94 samples (56.4%), while only 12 (12.8%) and 3 (3.2%) were tested
positive by nested PCR and conventional PCR, respectively. In conclusion, this
LAMP assay is a specific, sensitive, and rapid method for the detection of A.
phagocytophilum in sheep.
Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), formerly known
as the human granulocytic ehrlichiosis agent (HGE-A), is the causative agent of
human granulocytic anaplasmosis (HGA) in human beings and tick-borne fever in
domestic ruminants and wild animals (Dumler et al., 2001). It is classified into genus
Anaplasma, along with Anaplasma ovis, Anaplasma bovis, Anaplasma marginale, and
Anaplasma platys. In humans, HGA is characterized by headache, fever, malaise,
myalgia, leukopenia, thrombocytopenia, and evidence of hepatic injury (Dumler et al.,
2005). Tick-borne fever is reported as a febrile disease of deer, cows, hares and sheep,
with clinical signs varying from undetectable illness to severe febrile disease
associated with opportunistic infections (Stuen, 2007; Stuen et al., 2010; Woldehiwet,
2010; Thomas et al., 2012).
The most frequently used method for diagnosing A. phagocytophilum infection
involves microscopic identification of morulae on stained blood smears. However,
morulae could not be observed in many cases subsequently confirmed by other
criteria (Lester et al., 2005). On the other hand, serological evaluations are often
negative in the early phase of the disease, as antibodies are typically absent during the
first week of infection. In addition, the results of serological tests may lead to
misinterpretation and misdiagnosis due to cross-reactions (Waner et al., 2001). With
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the development of molecular biology methods, polymerase chain reaction (PCR) and
nucleotide sequencing are now widely used for the detection of A. phagocytophilum
(Massung and Slater, 2003; Dumler and Brouqui, 2004; Hulínská et al., 2004).
Nevertheless, the utility of insensitive conventional PCR, time-consuming and easily
contaminated nested PCR, and highly sensitive real-time PCR is also limited in rural
areas in that they require specific, expensive instruments. Since the incidence of A.
phagocytophilum infection in China is quite high in rural areas, rapid and simple
diagnostic methods are urgently needed.
In 2000, Notomi et al. (2000) developed a novel method, called loop-mediated
isothermal amplification (LAMP), which is highly specific, sensitive, simple and can
generate up to 109 fold amplification in less than 1 hr under isothermal conditions (60
C to 65 C). Over the last decade, LAMP has been widely used in laboratories to detect
pathogens of medical and veterinary importance, plant parasitic diseases, and
genetically modified products (Fu et al., 2011). Additionally, 2 LAMP assays based on
msp2 and gltA gene for detection of A. phagocytophilum have been described
previously (Pan et al., 2011; Lee et al., 2012). In this study, we aimed to develop a
LAMP method for the rapid detection of A. phagocytophilum DNA targeting 16S
rRNA gene and to evaluate its applicability by testing field samples and comparing
with nested PCR and conventional PCR.
Positive sample of A. phagocytophilum was preserved in the Parasitology
Laboratory of Henan Agricultural University. pMD18T plasmids containing partial A.
phagocytophilum 16S rRNA gene were used in sensitivity test of the LAMP assay.
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While blood samples respectively positive for A. ovis, A. bovis, Theileria luwenshuni,
Babesia motasi and Schistosoma japonicum were used as controls in specificity
identification. Among the control samples, the S. japonicum-positive blood DNA was
a gift from Nanjing Medical University, and others were collected by colleagues in
our laboratory. Genomic DNA (gDNA) was extracted from 1.5 mL whole blood
samples using the Genomic DNA extraction kit (Sangon Biotech, Shanghai, China)
according to the manufacturer’s instructions. The concentration of DNA was
measured by NanoDrop spectrometry (Thermo Fisher Scientific, Waltham,
Massachusetts) and the amounts of total DNAs ranged from 50 μg to 100 μg, then the
concentration was adjusted to 50 μg/μL.
The primer set was designed based on the 16S rRNA gene of A.
phagocytophilum (NR_044762). The forward outer primer (F3), backward outer
primer (B3), forward inner primer (FIP), and backward inner primer (BIP) (Table I)
were designed using the Primer Explorer program (version 4)
(http://primerexplorer.jp/elamp4.0./index.html). The primers were synthesized by
Dingguo Biotech (Beijing, China).
LAMP was performed in a total of 25 μL reaction mixture containing 1.6 μM
each FIP and BIP primers, 0.2 μM each F3 and B3 primers, 20 mM Tris-HCl (pH 8.8),
10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, and 0.1% Tween 20, 1.4 mM each
deoxynucleoside triphosphate (dNTP), 0.8 M betaine (Sigma-Aldrich, Beijing, China),
1 μL of Bst DNA polymerase large fragment (8U/μL) (NEB, Ipswich, MA) and 2 μL
of template DNA. The reaction mixture was incubated at 65 C for 60 min using a
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conventional heating block and then heated at 80 C for 10 min to terminate the
reaction. The specificity of LAMP was examined by testing 2 μL of gDNA of A. bovis,
A. ovis, B. motasi, T. luwenshuni, and S. japonicum, as well as A. phagocytophilum
positive sheep DNA confirmed previously as positive control and double distilled
water as negative control. The sensitivity of the reaction was evaluated by measuring
the concentration of plasmids, and the corresponding copy number was calculated
using the method previously described (Lee et al., 2006). The DNA was diluted to
contain 500 copies/μL and then serially diluted 10-fold. Two microliters were used in
each reaction mixture when the sensitivity of the assay was evaluated.
The nested PCR was conducted using primers and reaction condition described
previously (Zhang et al., 2016). The conventional PCR was performed using the
primer pair F3/B3 shown in Table I. The PCR reaction mixture contained 10×PCR
buffer (2.5 μL), 10 mM of dNTPs, 2.5 pmol of each primer, 0.25 μL of Taq DNA
polymerase (5 U/μL) (Takara, Dalian, China), 1 μL of template DNA and double
distilled water to a final volume of 25 μL. The reaction mixture was pre-denatured at
94 C for 5 min and subjected to 35 cycles at 94 C for 30 sec, 55 C for 30 sec, and 72
C for 40 sec with a final extension at 72 C for 10 min.
LAMP, nested and conventional PCR products were analyzed by electrophoresis
on a 1.5% agarose gel containing 1% DNA Green (TIANDZ, Beijing, China),
followed by visualization under UV light. Amplification of DNA in the LAMP
reaction was also monitored through direct visual inspection after addition of 1 μL
1/10 diluted SYBR green I (Invitrogen, Carlsbad, California). Moreover, the reaction
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mixture containing SYBR green I can also be visualized under a UV transilluminator.
To evaluate the feasibility of the LAMP assay for detection of A.
phagocytophilum in field samples, 94 sheep/goats blood samples collected from 5
farms in Henan province and 2 farms in Yunnan province were tested (The origin of
these blood samples are shown in Table SI). Sampling was approved by the Ethical
Committee of Henan Agricultural University, China. Genomic DNA was extracted
from the blood samples as described above. The samples were subjected to LAMP,
nested PCR and conventional PCR assay, and then the results were compared.
The optimal incubation temperature for LAMP with the A. phagocytophilum
primer set was established using temperatures ranging from 60 C to 65 C. All
temperatures gave positive results, finally 65 C was chosen as the reaction
temperature for all applications. A range of time periods for the reaction from 20 to
100 min were then tested at 65 C. And the results indicated that the most appropriate
incubation time was 60 min. The optimal concentration of MgSO4, dNTPs and betaine
was 8 mM, 1.4 mM and 0.6 M, respectively. Under these conditions, the set of 4
primers produced LAMP amplicons from DNA of A. phagocytophilum isolates in a
ladder pattern, while there were no products in negative control (double distilled
water) (Fig. 1). Upon addition of SYBR green I to the reaction mixtures, positive
reaction turned green whilst the negative remained orange. Furthermore, positive
reactions presented bright fluorescence under UV light while the negative just had
little (data not shown).
The sensitivity of the LAMP assay was determined by testing 10-fold serial
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dilutions of plasmids containing the 16S rRNA gene of A. phagocytophilum. The
detection limit of LAMP was 1 copy, compared with that of 1,000 copies when using
conventional PCR (Fig. 2), indicating that under the conditions used, the LAMP assay
was much more sensitive than conventional PCR for detection of A. phagocytophilum.
Specificity of the LAMP was proved by using genomic templates containing the
DNA of A. phagocytophilum, A. ovis, A. bovis, T. luwenshuni, B. motasi and S.
japonicum, respectively. Only DNA of A. phagocytophilum isolate tested positive,
while other species/parasites did not (Fig. 3). The results manifested that the LAMP
assay could differentiate A. phagocytophilum, A. ovis, A. bovis, T. luwenshuni, B.
motasi and S. japonicum, and there were no false-positive amplifications, using these
heterogonous species as templates and the reaction is specific for A. phagocytophilum.
A total of 94 sheep blood DNA samples were tested using the LAMP assay,
nested PCR and conventional PCR methods. With LAMP assay, 53 of the 94 DNA
samples (56.4%) were tested positive, while only 12 (12.8%) and 3 (3.2%) were
tested positive by nested PCR and conventional PCR, respectively (Table II).
Furthermore, no sample that tested positive by conventional PCR and nested PCR
tested negative by LAMP.
Anaplasma phagocytophilum has been known to cause diseases in domestic
ruminants and mammalian, as well as humans for decades (Dumler et al., 2005). The
greatest difficulty for this disease is not therapy but the diagnosis in the early phase of
infection. Otherwise, the A. phagocytophilum infection, often occurred in rural areas
where lack of rapid and sensitive diagnostic methods, may cause severe diseases and
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fatal outcomes (Lin et al., 2010; Yang et al., 2013, 2015). During the initial phase of A.
phagocytophilum infection, serological diagnosis, particularly indirect
immunofluorescence assay (IFA) are commonly used, but often give negative results
(Comer et al., 1999). While PCR techniques including nested PCR, real-time PCR and
conventional PCR are not widely applied in diagnosing this disease in rural areas due
to the need of a high-precision thermal cycler. In the present study, the established
LAMP assay can be performed simply in a water bath within one hour, and gene
amplification could be visualized and confirmed by naked eye monitoring through
color change when SYBR Green I was added. Considering the advantages of rapid
amplification, simple operation and easy detection, the LAMP method may have
potential applications for clinical diagnosis as well as surveillance of A.
phagocytophilum infection in developing countries without requiring sophisticated
equipment.
We demonstrated that the LAMP primers specifically amplified A.
phagocytophilum but not A. ovis, A. bovis, T. luwenshuni, B. motasi and S. japonicum,
confirming the high specificity of LAMP for the diagnosis of A. phagocytophilum
infection. The obtained results were consistent with previous studies that the
specificity of LAMP method is really high in detecting various pathogens (Thekisoe
et al., 2007; Curtis et al., 2008; Nkouawa and Sako, 2009; Okada et al., 2010).
Real-time PCR (Drazenovich et al., 2006; Wang et al., 2011) and nested PCR
(Alberto and Sparagano, 2006; Luan et al., 2008) have been reported to be more
sensitive than LAMP assay for the detection of A. phagocytophilum. In this study, the
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LAMP assay we developed was able to detect levels of DNA as low as 1 copy, which
was 103 fold lower than conventional PCR. In addition, in the following comparative
evaluation of 94 field samples, the LAMP assay also generated much higher positive
rate (56.4%) than other two PCR methods (nested PCR, 12.8% and conventional PCR,
3.2%). The higher sensitivity of LAMP assay than conventional PCR has also been
demonstrated by several other groups (Iseki et al., 2007; Liu et al., 2008). This may be
due to the fact that the sensitivity of LAMP was less affected by DNA polymerase
inhibitors in clinical samples, such as the various biological substances than was PCR
(Grab et al., 2005; Kaneko et al., 2007). Nevertheless, the false positive results
produced by LAMP could always not be avoided, which requests more careful
operation and more strict partition manipulation as conducted in the present study.
The application of LAMP assays for detection of A. phagocytophilum in humans
and dogs has been described previously targeting msp2 and gltA gene, respectively
(Pan et al., 2011; Lee et al., 2012). The detection limit of LAMP amplifying msp2
gene in the former study was 25 copies, which was 25-fold lower than conventional
PCR (625 copies); while in the latter investigation targeting gltA gene, the
sensitivities of LAMP and nested PCR methods were equivalent. Whilst in this study,
we developed the LAMP assay to detect A. phagocytophilum targeting 16S rRNA
gene in sheep. It is suggested that the similarity of the 16S rRNA gene could reach at
least 95% for the identification at the genus level and as high as 99% for the
identification at the species level (Fry et al., 1991; Clarridge, 2004). More importantly,
several investigations have declared that the amplification efficiency of 16S rRNA
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gene is much higher than coding genes, such as msp2, ankA, HSP70, groEL, etc
(Massung and Slater, 2003; Yang et al., 2016). This may explain why the LAMP assay
in the present study had a higher level of sensitivity than other reported LAMP
methods.
In conclusion, the LAMP assay targeting 16S rRNA gene we developed is simple,
rapid, sensitive, and specific for determining A. phagocytophilumin infection. For it is
fast and sensitive in testing clinical samples without sophisticated equipment and
complicated operation, it could be widely employed for the diagnosis of A.
phagocytophilum infection in the field and in rural areas of China where resource is
limited.
This work was supported by Earmarked Fund for China Modern Agro-industry
Technology Research System (nycytx-39). We thank Prof. Zhang from Nanjing
Medical University for kindly presenting the S. japonicum-positive blood DNA.
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Figure 1. Agarose gel electrophoresis of the LAMP products. 1, 2: Anaplasma
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phagocytophilum positive DNA; N: Negative control.
Figure 2. Detection limit of LAMP and conventional PCR. (A) The results of LAMP;
(B) The results of conventional PCR. 1-6: Anaplasma phagocytophilum positive DNA,
1,000 copies/μL, 100 copies/μL, 10 copies/μL, 1 copy/μL, 0.1 copy/μL, 0.01 copy/μL,
respectively; 7: Negative control.
Figure 3. The results of LAMP specificity test. Lanes 1-3: positive control
(Anaplasma phagocytophilum positive samples preserved in our laboratory); 4:
Anaplasma phagocytophilum; 5: Anaplasma bovis; 6: Anaplasma ovis; 7: Theleria
luwenshuni; 8: Babesia motasi; 9: Schistosoma japonicum; 10: dd H2O.
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