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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 1 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 2 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. 3 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 4 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 5 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 6 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 7 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 8 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 9 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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Journal of Medical Microbiology 62: 813–822. Yang, J., Z. Liu, Q. Niu, J. Liu, J. Xie, Q. Chen, Z. Chen, G. Guan, G. Liu, and J. Luo. 2016. Evaluation of different nested PCRs for detection of Anaplasma phagocytophilum in ruminants and ticks. Bmc Veterinary Research 12: 1-6. Zhang, Y., T. Li, Y. Cui, J. Wang, Y. Lv, R. Wang, F. Jian, L. Zhang, J. Wang, and G. Yang. 2016. The first report of Anaplasma phagocytophilum and a novel Theileria spp. co-infection in a South African giraffe. Parasitology International 65: 347-351. Figure 1. Agarose gel electrophoresis of the LAMP products. 1, 2: Anaplasma 15 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. 16