Accepted Manuscript A rapid, simple and sensitive loop-mediated isothermal amplification method to detect Anaplasma bovis in sheep and goats samples
Jinhong Wang, Yan Zhang, Yanyan Cui, Yaqun Yan, Xiaoxing Wang, Rongjun Wang, Fuchun Jian, Longxian Zhang, Changshen Ning PII: DOI: Reference:
S1383-5769(16)30568-2 doi: 10.1016/j.parint.2017.03.005 PARINT 1659
To appear in:
Parasitology International
Received date: Revised date: Accepted date:
30 December 2016 17 February 2017 24 March 2017
Please cite this article as: Jinhong Wang, Yan Zhang, Yanyan Cui, Yaqun Yan, Xiaoxing Wang, Rongjun Wang, Fuchun Jian, Longxian Zhang, Changshen Ning , A rapid, simple and sensitive loop-mediated isothermal amplification method to detect Anaplasma bovis in sheep and goats samples. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Parint(2017), doi: 10.1016/ j.parint.2017.03.005
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A rapid, simple and sensitive loop-mediated isothe rmal amplification method to detect
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Anaplasma bovis in sheep and goats samples
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Jinhong Wang # , Yan Zhang # , Yanyan Cui, Yaqun Yan, Xiaoxing Wang, Rongjun Wang,
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Fuchun Jian, Longxian Zhang, Changshen Ning*
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Parasitology Laboratory, College of Animal Science and Veterinary Medicine, Henan
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Agricultural University (all authors), Zhengzhou, P. R. China.
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The two authors contributed equally to this work.
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Corresponding author at:
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Prof. Changshen Ning
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College of Animal Science and Veterinary Medicine
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Henan Agricultural University
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Zhengzhou 450002, P. R. China
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Tel: +86 371 63555368
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Fax: +86 371 63558180
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E-mail: [email protected]
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Abstract 1
ACCEPTED MANUSCRIPT A loop-mediated isothermal amplification (LAMP) technique has been widely used in
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detecting the nucleic acid of various pathogenic bacteria. In this study, a set of four LAMP
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primers was designed to specifically test Anaplasma bovis. The LAMP assay was performed
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at 62°C for 60 min in a water bath. The specificity was confirmed by amplifying A. bovis
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isolate, while no cross reaction was observed with other five pathogens (Anaplasma ovis,
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Anaplasma phagocytophilum, Theileria luwenshuni, Babesia motasi and Schistosoma
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japonicum). The sensitivity of LAMP was 5×10 0 copies/μL, 100 times more than that of
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conventional PCR (5×102 copies/μL). Of 120 blood DNA extracted from sheep and goats
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field samples, 81 (67.5%), 22 (18.3%) and 43 (35.8%) were positively detected by LAMP,
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conventional PCR and nested PCR, respectively. The findings indicated that the developed
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LAMP assay is a new convenient tool for rapid and cost-effective detection of A. bovis.
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Keywords
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Anaplasma bovis; LAMP assay; 16S rRNA
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Anaplasma bovis, a member of the genus Anaplasma, which is the causative agent of bovine 2
ACCEPTED MANUSCRIPT anaplasmosis that causes fever, anemia, and weight loss in cattle throughout the tropical and
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subtropical regions of the world and found exclusively within parasitophorous vacuoles in the
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monocytes of domestic animals and wildlife. Since the first report of A. bovis infection in
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dogs in Brazil [1], a large number of research groups have indicated the presence of A. bovis
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in other animals (rabbit, tick, deer and cattle) [2-6]. The infection rates of A. bovis were 42.7
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and 23.8% in sheep and goats, respectively in the South Mediterranean area of Tunisia [7]. Ge
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et al. and Zhang et al. also reported the presence of A. bovis in sheep and goats blood samples
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in China [8, 9].
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In recent years, nucleic acid sequence-based amplification has been commonly employed in
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the detection of a wide range of pathogens. However, due to the need for expensive
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equipment and relatively high cost of real- time PCR, the lower sensitivity of conventional
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PCR [5] and the complicated procedure and time-consuming nested PCR [10], they all have
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not been widely utilized in many actual diagnostic applications. In 2000, a powerful
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innovative gene amplification technique named loop mediated isothermal amplification
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(LAMP) was developed for early detection and identification of microbial diseases [11]. The
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whole procedure of LAMP assay is very simple and rapid whe rein the amplification can be
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finished in less than 1 h by incubating all the reagents in a single tube under isothermal
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conditions employing a set of four specially designed primers spanning six distinct sequences
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of the target gene [12]. The high specificity of LAMP were verified by several studies [13,14],
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this is attributable to recognition of target sequence by six independent sequences in the initial
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stage and by four independent sequences during the later stages [11]. Moreover, LAMP
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products can be easily visualized by gel electrophoresis or by measuring turbidity caused by a
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white precipitate of magnesium pyrophosphate [11, 15]. With these advantages, LAMP could
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be widely used as a simple and rapid diagnostic tool at the early stage of diseases in remote
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areas where lack of expensive equipments.
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A. bovis was found in 49.6% of 262 goat blood samples by using nested PCR methods [16].
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While DNA fragments of A. bovis was also detected in cattle on Yonaguni Island from Japan
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by employing nested PCR [5]. In the present study, a LAMP assay targeting 16S rRNA gene
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was established for detecting A. bovis, followed by field samples validation of the method
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with sheep and goat blood samples.
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This study was conducted in accordance with the Chinese Laboratory Animal Administration
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Act of 1988. This research protocol was reviewed and approved by the Research Committee
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of Henan Agricultural University. The field studies did not involve endangered or protected
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species. A total of 120 whole blood samples were collected from small ruminants including 84
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goats from Luoyang, Pingdingshan, Jiyuan and Xinmi cities of Henan province, and Longling
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county of Yunnan province, 36 sheep blood specimens originated from Zhengzhou, Jiaozuo
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and Luoyang cities of Henan province between April and October in 2013. The blood samples
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were taken from the jugular vein of each animal and collected in a sterile tube containing an
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anticoagulant (EDTA). Genomic DNA (gDNA) was extracted from 500 μL of each sample
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using a Blood Genomic DNA Extraction kit (Tiangen, Beijing, China) according to the
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manufacturer’s instructions, and the concentration of DNA was determined by NanoDrop
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ACCEPTED MANUSCRIPT spectrometry (Thermo Fisher Scientific, MA, USA). A. bovis-positive blood samples
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preserved in the parasitology laboratory of Henan Agricultural University were used to
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establish and optimize the reaction condition of the LAMP assay. Blood sample positive for
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Schistosoma japonicum was given as a present by professor Zhang from Nanjing Medical
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University. While other four pathogens (A. ovis, A. phagocytophilum, Theileria luwenshuni,
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and Babesia motasi) –positive DNA were collected by our colleagues and preserved in this
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laboratory.
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The forward outer primer (F3), backward outer primer (B3), forward inner primer (FIP), and
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backward inner primer (BIP) were designed using the online primer design software Primer
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Explorer V4 (http://primerexplorer.jp/elamp4.0.0/index.html), on the basis of A. bovis 16S
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rRNA gene sequence FJ169957 after multiple sequences alignment. The binding positions of
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each primer (Fig.1) and the sequence of the primers were shown in Table 1. LAMP was
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performed in a total of 25 μL reaction mixture containing 1.6 μM each FIP and BIP primers,
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0.2 μM each F3 and B3 primers, 20 mM Tris-HCl (pH8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 8
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mM MgSO4 , and 0.1% Tween 20, 1.4 mM each deoxynucleoside triphosphate (dNTP), 0.8 M
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betaine (Sigma-Aldrich, Beijing, China), 8 U of Bst DNA polymerase large fragment (New
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England BioLabs Inc., United Kingdom) and 2 μL of template DNA. The reaction mixture
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was incubated at 62 °C for 60 min using a conventional heating block and then heated at
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80 °C for 10 min to terminate the reaction. The specificity of LAMP was examined by testing
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5 μL of gDNA of A. phagocytophilum, A. bovis, A. ovis, B. motasi, T. luwenshuni, the S.
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japonicum, as well as A. bovis positive sheep DNA confirmed previously as positive control
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ACCEPTED MANUSCRIPT and double distilled water as negative control. The sensitivity of the reaction was evaluated by
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measuring the concentration of DNA, and the corresponding copy number was calculated
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using the method previously described [17]. The DNA was diluted to contain 500 copies/μL
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and then serially diluted 10- fold. Two micro liters were used in each reaction mixture when
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the sensitivity of the assay was evaluated.
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Nested PCR was conducted using primers and reaction condition described previously [18].
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The conventional PCR was performed using the primer pair F3/B3 shown in Table 1,
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generating a product of 236 bp. The reaction mixture, condition and procedure were
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performed as the second round PCR in a previous study [18]. LAMP, nested and conventional
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PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing 1.0% DNA
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Green (TIANDZ, Beijing, China), followed by visualization under UV light. Amplification of
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DNA in the LAMP reaction was also monitored through direct visual inspection after addition
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of 1.0 μL 1/10 diluted SYBR green I (Invitrogen, USA). What’s more, the reaction mixture
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containing SYBR green I can also be visualized under a UV transilluminator.
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The 120 whole blood DNA samples were used to validate the clinical application of the
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LAMP assay, while nested PCR and conventional PCR were also utilized to compare the
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positive rate in clinical samples with the LAMP.
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A range of temperatures from 60~65℃ were explored to find out the optimal reaction
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temperature of LAMP, and the results ( data not shown) manifested that characteristic gradient 6
ACCEPTED MANUSCRIPT amplification bands with highest brightness was observed at 62℃, which was chosen as the
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optimal reaction temperature for all applications. Then time periods from 30 min to 90 min
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for the reaction were tested at this temperature. Analysis of the results indicated that
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incubation for 40 min was sufficient to allow positive bands to occur. And the optimal
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concentration of MgSO 4 , dNTPs and betaine was 8 mM, 1.4 mM and 0.6 M, respectively.
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Under these conditions, the LAMP primers produced amplicons from genomic DNA positive
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for A. bovis in a ladder pattern, while there were no products in negative control (Fig. 2A).
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After adding SYBR green I to the reaction mixture, positive samples turned green while
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negative remained orange (Fig. 2B). What’s more, positive reaction mixtures presented bright
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fluorescence under UV light whilst the negative just had little (Fig. 2C).
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The specificity of LAMP was evaluated by testing the genomic DNA positive for A. bovis, A.
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phagocytophilum, A. ovis, T. luwenshuni, B. motasi and S. japonicum, respectively. As shown
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in figure 3, only DNA of A. bovis isolate gave amplicon in a ladder pattern, which manifested
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that the LAMP assay had no false-positive results and also was specific for A. bovis. The
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sensitivity of LAMP was assessed by using 10- fold dilution of the DNA positive for A. bovis
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as the templates of amplifications. The results in figure 4 showed that the detection limit of
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conventional PCR was 5×102 copies/μL (Fig. 4B), while that of LAMP was 5×10 0 copies/μL
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(Fig. 4A), which had much higher sensitivity than the former.
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LAMP, conventional PCR and nested PCR were conducted to detected 120 sheep/goats blood
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DNA samples, with A. bovis positive rates 67.5% (81/120), 18.3% (22/120) and 35.8% 7
ACCEPTED MANUSCRIPT (43/120), respectively. Whether feeding pattern of the animals was grazing (57.0% vs 15.1%
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and 26.7%) or stabling (94.1% vs 26.5% and 58.8%) (Table 2), whether in sheep (77.8% vs
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16.7% and 41.7%) or goats (63.1% vs 19.0% and 33.3%), the LAMP method had highest
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positive rates. In the field-applied experiments against sheep and goats, the LAMP assay
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exhibited higher detection abilities for the target A. bovis infection than those of conventional
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PCR and nested PCR.
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In this study, we successfully developed a LAMP assay for rapid detection of A. bovis in a
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water bath at isothermal condition. A set of four primers was designed to amplify the target
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DNA fragment of 16S rRNA gene. Our findings regarding high specificity are in agreement
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with previous reports of other LAMP methods [13, 14]. In addition, the higher detection
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ability of the established LAMP assay was visible regardless of feeding pattern or breed of
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small ruminants (Table 2). Furthermore, the LAMP assay was highly sensitive, as DNA with
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as few as five copies per micro liter was detectable in LAMP reaction. Moreover, the reaction
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can be monitored with the naked eye by adding chromogenic agents, such as SYBR green I,
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which produces a color-transform from orange to green in natural light or presents bright
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fluorescence under UV light [14, 19], which were also used in the present study to distinguish
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the positive and negative reaction mixtures (Fig. 2).
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Recently, several groups have reported the infection of A. bovis in small ruminants. In 2012,
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Liu et al. found that 49.6% of 262 goat blood samples from central and southern China were
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to be positive for A. bovis [16], later in 2016, the same research group reported a similar 8
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collected from southeastern China [8], While in other studies concerning A. bovis infection in
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small ruminants, the occurrence were all lower than the former two reports, with positive rates
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of 19.2% in sheep and 25.1% in goats from six provinces of China [9] and 42.7% and 23.8%
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in sheep and goats, respectively in the South Mediterranean area of Tunisia [7]. Zhang et al.
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also reported that A. bovis could be detected in sheep and goats milk specimens using nested
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PCR methods [20].
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The positive rates of A. bovis obtained with nested PCR in the present study (41.7% in sheep
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and 33.3% in goats) were lower than those reported by Liu et al. (49.6% in goats) [16] and Ge
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et al. (49.0% in sheep and goats) [8] but higher than Zhang et al.’s report (19.2% in sheep and
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25.1% in goats) [9], while the positive rates of LAMP assay were much higher than all those
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PCR based methods. However, the prevalence of A. bovis in sheep and goats had no
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significant difference whether using LAMP assay or other two PCR methods, which was not
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consistent with the results observed by Ben Said et al. that A. bovis prevalence in goats was
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lower than that found in the investigated sheep (23.8% vs 42.7%) by nested PCR[7].
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Not surprisingly, the occurrence of A. bovis in grazing animals was higher than in stabling
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animals, no matter what the detection methods used (Table 2), this is attributed to the higher
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chances of biting by tick vectors in grazing animals than animals living in stocks. The similar
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results were also observed by Cui et al. in dogs under different feeding patterns that the
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Anaplasma incidence in stray dogs was much higher than in pet dogs (40.7% vs 4.0%) [21]. 9
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Few studies focus on the pathogenicity of A. bovis in small ruminants, except one report
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conducted in 2012, in which two 6 months-old sheep were inoculated with A. bovis positive
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field blood samples and inclusions were only found in the monocytes for just one day. The
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experimental animals developed either a short fever or no fever at all and recovered rapidly
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[16]. Although infection is often asymptomatic, A. bovis can cause a variety of clinical
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symptoms, including fever and reduced body weight and possibly death of naive or stressed
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cattle, which would bring great loss to animal industry.
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In conclusion, our LAMP method is a new, convenient tool for identifying A. bovis. In the
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present study, positive reactions could be achieved with the LAMP assay at 62 ℃, which
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strongly suggests that the method can be widely applicable in rural areas where lack of
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expensive facilities, for routine screening of A. bovis infection.
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Acknowledgments
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This work was supported by Earmarked Found for China Modern Agro- industry Technology
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Research System (nycytx-39). We thank the farmers in Henan and Yunnan for their
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cooperation. We thank Prof. Zhang from Nanjing Medical University for kindly presenting
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blood sample positive for S. japonicum.
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Table 1 The sequences of A. bovis LAMP primers 12
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Primer Name
Primer sequence（5′-3′）
Primer length
F3
GGGCATGTAGGTGGTTTGG
19 bp
B3
GCGTGGACTACCAGGGTAT
19 bp
FIP
TCTCCCGGACTCCAGTCTGGTAAAGGTGAAATGCCAGG GC
40 bp
BIP
ATTAGGAGGAACACCAGTGGCGCACGCTTTCGCACCTC
40 bp
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Positive rates of
Conventional PCR
Stabling
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13/86(15.1)
Grazing
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9/34(26.5)
Sheep Goats
36 84
6/36(16.7) 16/84(19.0)
Total
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22/120(18.3)
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No. sampled
Nested-PCR
LAMP
23/86(26.7)
49/86(57.0)
20/34(58.8)
32/34(94.1)
15/36(41.7) 28/84(33.3)
28/36(77.8) 53/84(63.1)
43/120(35.8)
81/120(67.5)
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Feeding pattern/Breed
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Table 2 The detection results of field samples using different methods
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Figure legends
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Figure 1. The binding positions of LAMP primers on the 16S rRNA gene sequence of A.
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bovis (FJ169957). The direction of the arrows is 5′-3′.
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Figure
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A: Agarose gel electrophoresis of the LAMP products; B: LAMP products adding SYBR
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green I were observed by naked eye; C: LAMP reaction mixtures containing SYBR green I
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were visualized under UV light. A/B/C: 1-7: A. bovis positive DNA; N: Negative control. M:
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DL2000 DNA Marker.
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Figure 3. The result of LAMP specificity test. M: DL2000 DNA Marker; 1-7: The
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amplification results of A. bovis, A. phagocytophilum, A. ovis, B. motasi, T. luwenshuni, S.
The
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Figure 4. Detection limits of LAMP and conventional PCR. A: The results of LAMP assay; B:
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The results of conventional PCR. M: DL2000 DNA Marker; 1~7: A. bovis-positive DNA，the
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copy numbers, in turn, were 103 ~10-3 copies/μL; N: Negative control.
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Figure S1. Repeatability test of the LAMP assay. M: DL2000 DNA Marker; 1~3: three
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repeated tests of A. bovis-positive DNA sample 1; 4-6: three repeated tests of A. bovis-positive
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DNA sample 2; 7-9: Negative control.
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Jinhong Wang, Yan Zhang, Yanyan Cui, Yaqun Yan, Xiaoxing Wang, Rongjun Wang, Fuchun Jian, Longxian Zhang, Changshen Ning PII: DOI: Reference:
S1383-5769(16)30568-2 doi: 10.1016/j.parint.2017.03.005 PARINT 1659
To appear in:
Parasitology International
Received date: Revised date: Accepted date:
30 December 2016 17 February 2017 24 March 2017
Please cite this article as: Jinhong Wang, Yan Zhang, Yanyan Cui, Yaqun Yan, Xiaoxing Wang, Rongjun Wang, Fuchun Jian, Longxian Zhang, Changshen Ning , A rapid, simple and sensitive loop-mediated isothermal amplification method to detect Anaplasma bovis in sheep and goats samples. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Parint(2017), doi: 10.1016/ j.parint.2017.03.005
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A rapid, simple and sensitive loop-mediated isothe rmal amplification method to detect
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Anaplasma bovis in sheep and goats samples
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Jinhong Wang # , Yan Zhang # , Yanyan Cui, Yaqun Yan, Xiaoxing Wang, Rongjun Wang,
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Fuchun Jian, Longxian Zhang, Changshen Ning*
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Parasitology Laboratory, College of Animal Science and Veterinary Medicine, Henan
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Agricultural University (all authors), Zhengzhou, P. R. China.
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The two authors contributed equally to this work.
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#
Corresponding author at:
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Prof. Changshen Ning
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College of Animal Science and Veterinary Medicine
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Henan Agricultural University
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Zhengzhou 450002, P. R. China
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Tel: +86 371 63555368
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Fax: +86 371 63558180
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E-mail: [email protected]
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Abstract 1
ACCEPTED MANUSCRIPT A loop-mediated isothermal amplification (LAMP) technique has been widely used in
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detecting the nucleic acid of various pathogenic bacteria. In this study, a set of four LAMP
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primers was designed to specifically test Anaplasma bovis. The LAMP assay was performed
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at 62°C for 60 min in a water bath. The specificity was confirmed by amplifying A. bovis
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isolate, while no cross reaction was observed with other five pathogens (Anaplasma ovis,
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Anaplasma phagocytophilum, Theileria luwenshuni, Babesia motasi and Schistosoma
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japonicum). The sensitivity of LAMP was 5×10 0 copies/μL, 100 times more than that of
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conventional PCR (5×102 copies/μL). Of 120 blood DNA extracted from sheep and goats
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field samples, 81 (67.5%), 22 (18.3%) and 43 (35.8%) were positively detected by LAMP,
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conventional PCR and nested PCR, respectively. The findings indicated that the developed
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LAMP assay is a new convenient tool for rapid and cost-effective detection of A. bovis.
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Keywords
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Anaplasma bovis; LAMP assay; 16S rRNA
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Anaplasma bovis, a member of the genus Anaplasma, which is the causative agent of bovine 2
ACCEPTED MANUSCRIPT anaplasmosis that causes fever, anemia, and weight loss in cattle throughout the tropical and
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subtropical regions of the world and found exclusively within parasitophorous vacuoles in the
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monocytes of domestic animals and wildlife. Since the first report of A. bovis infection in
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dogs in Brazil [1], a large number of research groups have indicated the presence of A. bovis
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in other animals (rabbit, tick, deer and cattle) [2-6]. The infection rates of A. bovis were 42.7
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and 23.8% in sheep and goats, respectively in the South Mediterranean area of Tunisia [7]. Ge
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et al. and Zhang et al. also reported the presence of A. bovis in sheep and goats blood samples
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in China [8, 9].
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In recent years, nucleic acid sequence-based amplification has been commonly employed in
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the detection of a wide range of pathogens. However, due to the need for expensive
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equipment and relatively high cost of real- time PCR, the lower sensitivity of conventional
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PCR [5] and the complicated procedure and time-consuming nested PCR [10], they all have
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not been widely utilized in many actual diagnostic applications. In 2000, a powerful
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innovative gene amplification technique named loop mediated isothermal amplification
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(LAMP) was developed for early detection and identification of microbial diseases [11]. The
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whole procedure of LAMP assay is very simple and rapid whe rein the amplification can be
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finished in less than 1 h by incubating all the reagents in a single tube under isothermal
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conditions employing a set of four specially designed primers spanning six distinct sequences
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of the target gene [12]. The high specificity of LAMP were verified by several studies [13,14],
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this is attributable to recognition of target sequence by six independent sequences in the initial
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stage and by four independent sequences during the later stages [11]. Moreover, LAMP
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products can be easily visualized by gel electrophoresis or by measuring turbidity caused by a
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white precipitate of magnesium pyrophosphate [11, 15]. With these advantages, LAMP could
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be widely used as a simple and rapid diagnostic tool at the early stage of diseases in remote
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areas where lack of expensive equipments.
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A. bovis was found in 49.6% of 262 goat blood samples by using nested PCR methods [16].
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While DNA fragments of A. bovis was also detected in cattle on Yonaguni Island from Japan
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by employing nested PCR [5]. In the present study, a LAMP assay targeting 16S rRNA gene
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was established for detecting A. bovis, followed by field samples validation of the method
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with sheep and goat blood samples.
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This study was conducted in accordance with the Chinese Laboratory Animal Administration
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Act of 1988. This research protocol was reviewed and approved by the Research Committee
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of Henan Agricultural University. The field studies did not involve endangered or protected
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species. A total of 120 whole blood samples were collected from small ruminants including 84
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goats from Luoyang, Pingdingshan, Jiyuan and Xinmi cities of Henan province, and Longling
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county of Yunnan province, 36 sheep blood specimens originated from Zhengzhou, Jiaozuo
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and Luoyang cities of Henan province between April and October in 2013. The blood samples
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were taken from the jugular vein of each animal and collected in a sterile tube containing an
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anticoagulant (EDTA). Genomic DNA (gDNA) was extracted from 500 μL of each sample
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using a Blood Genomic DNA Extraction kit (Tiangen, Beijing, China) according to the
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manufacturer’s instructions, and the concentration of DNA was determined by NanoDrop
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ACCEPTED MANUSCRIPT spectrometry (Thermo Fisher Scientific, MA, USA). A. bovis-positive blood samples
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preserved in the parasitology laboratory of Henan Agricultural University were used to
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establish and optimize the reaction condition of the LAMP assay. Blood sample positive for
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Schistosoma japonicum was given as a present by professor Zhang from Nanjing Medical
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University. While other four pathogens (A. ovis, A. phagocytophilum, Theileria luwenshuni,
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and Babesia motasi) –positive DNA were collected by our colleagues and preserved in this
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laboratory.
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The forward outer primer (F3), backward outer primer (B3), forward inner primer (FIP), and
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backward inner primer (BIP) were designed using the online primer design software Primer
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Explorer V4 (http://primerexplorer.jp/elamp4.0.0/index.html), on the basis of A. bovis 16S
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rRNA gene sequence FJ169957 after multiple sequences alignment. The binding positions of
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each primer (Fig.1) and the sequence of the primers were shown in Table 1. LAMP was
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performed in a total of 25 μL reaction mixture containing 1.6 μM each FIP and BIP primers,
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0.2 μM each F3 and B3 primers, 20 mM Tris-HCl (pH8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 8
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mM MgSO4 , and 0.1% Tween 20, 1.4 mM each deoxynucleoside triphosphate (dNTP), 0.8 M
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betaine (Sigma-Aldrich, Beijing, China), 8 U of Bst DNA polymerase large fragment (New
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England BioLabs Inc., United Kingdom) and 2 μL of template DNA. The reaction mixture
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was incubated at 62 °C for 60 min using a conventional heating block and then heated at
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80 °C for 10 min to terminate the reaction. The specificity of LAMP was examined by testing
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5 μL of gDNA of A. phagocytophilum, A. bovis, A. ovis, B. motasi, T. luwenshuni, the S.
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japonicum, as well as A. bovis positive sheep DNA confirmed previously as positive control
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ACCEPTED MANUSCRIPT and double distilled water as negative control. The sensitivity of the reaction was evaluated by
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measuring the concentration of DNA, and the corresponding copy number was calculated
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using the method previously described [17]. The DNA was diluted to contain 500 copies/μL
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and then serially diluted 10- fold. Two micro liters were used in each reaction mixture when
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the sensitivity of the assay was evaluated.
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Nested PCR was conducted using primers and reaction condition described previously [18].
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The conventional PCR was performed using the primer pair F3/B3 shown in Table 1,
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generating a product of 236 bp. The reaction mixture, condition and procedure were
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performed as the second round PCR in a previous study [18]. LAMP, nested and conventional
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PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing 1.0% DNA
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Green (TIANDZ, Beijing, China), followed by visualization under UV light. Amplification of
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DNA in the LAMP reaction was also monitored through direct visual inspection after addition
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of 1.0 μL 1/10 diluted SYBR green I (Invitrogen, USA). What’s more, the reaction mixture
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containing SYBR green I can also be visualized under a UV transilluminator.
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The 120 whole blood DNA samples were used to validate the clinical application of the
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LAMP assay, while nested PCR and conventional PCR were also utilized to compare the
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positive rate in clinical samples with the LAMP.
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A range of temperatures from 60~65℃ were explored to find out the optimal reaction
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temperature of LAMP, and the results ( data not shown) manifested that characteristic gradient 6
ACCEPTED MANUSCRIPT amplification bands with highest brightness was observed at 62℃, which was chosen as the
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optimal reaction temperature for all applications. Then time periods from 30 min to 90 min
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for the reaction were tested at this temperature. Analysis of the results indicated that
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incubation for 40 min was sufficient to allow positive bands to occur. And the optimal
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concentration of MgSO 4 , dNTPs and betaine was 8 mM, 1.4 mM and 0.6 M, respectively.
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Under these conditions, the LAMP primers produced amplicons from genomic DNA positive
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for A. bovis in a ladder pattern, while there were no products in negative control (Fig. 2A).
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After adding SYBR green I to the reaction mixture, positive samples turned green while
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negative remained orange (Fig. 2B). What’s more, positive reaction mixtures presented bright
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fluorescence under UV light whilst the negative just had little (Fig. 2C).
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The specificity of LAMP was evaluated by testing the genomic DNA positive for A. bovis, A.
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phagocytophilum, A. ovis, T. luwenshuni, B. motasi and S. japonicum, respectively. As shown
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in figure 3, only DNA of A. bovis isolate gave amplicon in a ladder pattern, which manifested
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that the LAMP assay had no false-positive results and also was specific for A. bovis. The
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sensitivity of LAMP was assessed by using 10- fold dilution of the DNA positive for A. bovis
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as the templates of amplifications. The results in figure 4 showed that the detection limit of
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conventional PCR was 5×102 copies/μL (Fig. 4B), while that of LAMP was 5×10 0 copies/μL
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(Fig. 4A), which had much higher sensitivity than the former.
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LAMP, conventional PCR and nested PCR were conducted to detected 120 sheep/goats blood
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DNA samples, with A. bovis positive rates 67.5% (81/120), 18.3% (22/120) and 35.8% 7
ACCEPTED MANUSCRIPT (43/120), respectively. Whether feeding pattern of the animals was grazing (57.0% vs 15.1%
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and 26.7%) or stabling (94.1% vs 26.5% and 58.8%) (Table 2), whether in sheep (77.8% vs
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16.7% and 41.7%) or goats (63.1% vs 19.0% and 33.3%), the LAMP method had highest
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positive rates. In the field-applied experiments against sheep and goats, the LAMP assay
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exhibited higher detection abilities for the target A. bovis infection than those of conventional
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PCR and nested PCR.
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In this study, we successfully developed a LAMP assay for rapid detection of A. bovis in a
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water bath at isothermal condition. A set of four primers was designed to amplify the target
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DNA fragment of 16S rRNA gene. Our findings regarding high specificity are in agreement
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with previous reports of other LAMP methods [13, 14]. In addition, the higher detection
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ability of the established LAMP assay was visible regardless of feeding pattern or breed of
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small ruminants (Table 2). Furthermore, the LAMP assay was highly sensitive, as DNA with
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as few as five copies per micro liter was detectable in LAMP reaction. Moreover, the reaction
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can be monitored with the naked eye by adding chromogenic agents, such as SYBR green I,
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which produces a color-transform from orange to green in natural light or presents bright
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fluorescence under UV light [14, 19], which were also used in the present study to distinguish
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the positive and negative reaction mixtures (Fig. 2).
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Recently, several groups have reported the infection of A. bovis in small ruminants. In 2012,
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Liu et al. found that 49.6% of 262 goat blood samples from central and southern China were
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to be positive for A. bovis [16], later in 2016, the same research group reported a similar 8
ACCEPTED MANUSCRIPT results with the prevalence of A. bovis was 49.0% in 100 sheep and goats blood samples
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collected from southeastern China [8], While in other studies concerning A. bovis infection in
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small ruminants, the occurrence were all lower than the former two reports, with positive rates
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of 19.2% in sheep and 25.1% in goats from six provinces of China [9] and 42.7% and 23.8%
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in sheep and goats, respectively in the South Mediterranean area of Tunisia [7]. Zhang et al.
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also reported that A. bovis could be detected in sheep and goats milk specimens using nested
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PCR methods [20].
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The positive rates of A. bovis obtained with nested PCR in the present study (41.7% in sheep
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and 33.3% in goats) were lower than those reported by Liu et al. (49.6% in goats) [16] and Ge
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et al. (49.0% in sheep and goats) [8] but higher than Zhang et al.’s report (19.2% in sheep and
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25.1% in goats) [9], while the positive rates of LAMP assay were much higher than all those
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PCR based methods. However, the prevalence of A. bovis in sheep and goats had no
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significant difference whether using LAMP assay or other two PCR methods, which was not
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consistent with the results observed by Ben Said et al. that A. bovis prevalence in goats was
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lower than that found in the investigated sheep (23.8% vs 42.7%) by nested PCR[7].
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Not surprisingly, the occurrence of A. bovis in grazing animals was higher than in stabling
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animals, no matter what the detection methods used (Table 2), this is attributed to the higher
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chances of biting by tick vectors in grazing animals than animals living in stocks. The similar
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results were also observed by Cui et al. in dogs under different feeding patterns that the
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Anaplasma incidence in stray dogs was much higher than in pet dogs (40.7% vs 4.0%) [21]. 9
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Few studies focus on the pathogenicity of A. bovis in small ruminants, except one report
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conducted in 2012, in which two 6 months-old sheep were inoculated with A. bovis positive
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field blood samples and inclusions were only found in the monocytes for just one day. The
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experimental animals developed either a short fever or no fever at all and recovered rapidly
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[16]. Although infection is often asymptomatic, A. bovis can cause a variety of clinical
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symptoms, including fever and reduced body weight and possibly death of naive or stressed
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cattle, which would bring great loss to animal industry.
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In conclusion, our LAMP method is a new, convenient tool for identifying A. bovis. In the
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present study, positive reactions could be achieved with the LAMP assay at 62 ℃, which
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strongly suggests that the method can be widely applicable in rural areas where lack of
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expensive facilities, for routine screening of A. bovis infection.
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Acknowledgments
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This work was supported by Earmarked Found for China Modern Agro- industry Technology
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Research System (nycytx-39). We thank the farmers in Henan and Yunnan for their
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cooperation. We thank Prof. Zhang from Nanjing Medical University for kindly presenting
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blood sample positive for S. japonicum.
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Table 1 The sequences of A. bovis LAMP primers 12
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Primer Name
Primer sequence（5′-3′）
Primer length
F3
GGGCATGTAGGTGGTTTGG
19 bp
B3
GCGTGGACTACCAGGGTAT
19 bp
FIP
TCTCCCGGACTCCAGTCTGGTAAAGGTGAAATGCCAGG GC
40 bp
BIP
ATTAGGAGGAACACCAGTGGCGCACGCTTTCGCACCTC
40 bp
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Positive rates of
Conventional PCR
Stabling
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13/86(15.1)
Grazing
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9/34(26.5)
Sheep Goats
36 84
6/36(16.7) 16/84(19.0)
Total
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22/120(18.3)
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No. sampled
Nested-PCR
LAMP
23/86(26.7)
49/86(57.0)
20/34(58.8)
32/34(94.1)
15/36(41.7) 28/84(33.3)
28/36(77.8) 53/84(63.1)
43/120(35.8)
81/120(67.5)
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Feeding pattern/Breed
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Table 2 The detection results of field samples using different methods
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Figure legends
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Figure 1. The binding positions of LAMP primers on the 16S rRNA gene sequence of A.
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bovis (FJ169957). The direction of the arrows is 5′-3′.
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Figure
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A: Agarose gel electrophoresis of the LAMP products; B: LAMP products adding SYBR
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green I were observed by naked eye; C: LAMP reaction mixtures containing SYBR green I
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were visualized under UV light. A/B/C: 1-7: A. bovis positive DNA; N: Negative control. M:
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DL2000 DNA Marker.
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Figure 3. The result of LAMP specificity test. M: DL2000 DNA Marker; 1-7: The
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amplification results of A. bovis, A. phagocytophilum, A. ovis, B. motasi, T. luwenshuni, S.
The
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Figure 4. Detection limits of LAMP and conventional PCR. A: The results of LAMP assay; B:
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The results of conventional PCR. M: DL2000 DNA Marker; 1~7: A. bovis-positive DNA，the
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copy numbers, in turn, were 103 ~10-3 copies/μL; N: Negative control.
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Figure S1. Repeatability test of the LAMP assay. M: DL2000 DNA Marker; 1~3: three
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repeated tests of A. bovis-positive DNA sample 1; 4-6: three repeated tests of A. bovis-positive
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DNA sample 2; 7-9: Negative control.
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