- Email: [email protected]
Rapid diagnosis of Theileria annulata by recombinase polymerase amplification combined with a lateral flow strip (LF-RPA) in epidemic regions
Accepted Manuscript Title: Rapid diagnosis of Theileria annulata by recombinase polymerase amplification combined with a lateral flow strip (LF-RPA) in epidemic regions Authors: Fangyuan Yin, Junlong Liu, Aihong Liu, Youquan Li, Jianxun Luo, Guiquan Guan, Hong Yin PII: DOI: Reference:
S0304-4017(17)30070-5 http://dx.doi.org/doi:10.1016/j.vetpar.2017.02.019 VETPAR 8268
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
23-11-2016 14-2-2017 18-2-2017
Please cite this article as: Yin, Fangyuan, Liu, Junlong, Liu, Aihong, Li, Youquan, Luo, Jianxun, Guan, Guiquan, Yin, Hong, Rapid diagnosis of Theileria annulata by recombinase polymerase amplification combined with a lateral flow strip (LF-RPA) in epidemic regions.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2017.02.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Rapid diagnosis of Theileria annulata by recombinase polymerase amplification combined with a lateral flow strip (LF-RPA) in epidemic regions
Fangyuan Yin a, Junlong Liu a, Aihong Liu a, Youquan Li a, Jianxun Luo a, Guiquan Guan a*, Hong Yin a, b*
a
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of
Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Gansu 730046, P. R. China. b
Jiangsu Co-innovation Center for Prevention and Control of Important Animal
Infectious Diseases and Zoonoses, Yangzhou, 225009, P. R. China.
*Corresponding authors: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu 730046, P. R. China. E-mail: [email protected]; [email protected] Tel.: +86 931 8342681 Fax: +86 931 8340977
1
Highlights
A LF-RPA was firstly developed to detect Theileria annulata infections.
The sensitivity of LF-RPA is similar to PCR for detecting the field samples.
The LF-RPA could be used to surveil and control of Theileria annulata.
Abstract Rapid and accurate diagnosis of Theileria annulata infection contributes to the formulation of strategies to eradicate this parasite. A simple and efficient diagnostic tool, recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip, was used in detection of Theileria and compared to other methods that require expensive instruments and skilled personnel. Herein, we established and optimized an LF-RPA method to detect the cytochrome b gene of T. annulata mitochondrial DNA from experimentally infected and field-collected blood samples. This method has many unparalleled characteristics, including that it is rapid (clear detection in 5 min at constant temperature), sensitive (the limitation of detection is at least 2 pg genomic DNA), and specific (no cross-reaction with other piroplasms that infect cattle). The LF-RPA assay was evaluated via testing 17 field blood samples and comparing the results of that of a PCR, showing 100% agreement, which demonstrates the ability of the LF-RPA assay to detect T. annulata infections in small number of samples (n = 17). Taken together, the results indicate that this method could be used as an ideal 2
diagnostic tool for detecting T. annulata in endemic regions with limited to fewer and local resources and could also be a potential technique for the surveillance and control of blood protozoa.
Keywords: Theileria annulata; Diagnosis; Recombinase polymerase amplification; Lateral flow;
1. Introduction Theileria annulata, one of the tick-borne protozoan parasites infective to ruminants, is the pathogen of tropical theileriosis and causes great losses to the cattle industry worldwide. (Gharbi et al., 2006). Currently, diagnostic tests mainly consist of microscopic examinations (Ahmed et al., 2002), serological assays (Renneker et al., 2008) and PCR-based detection (Gomes and Inácio, 2015) for effective prevention and control of tropical theileriosis. However, these methods are not well suited for direct field testing because of their complicated operation processes, trained personnel and time consuming (Pai et al., 2012). It is essential to develop a rapid, simple and clinical applications test to improve the efficacy of current control programmes. Recombinase polymerase amplification is a novel isothermal amplification method that does not require sophisticated equipment or thermal denaturation and can be performed for less than 20 min at a low and constant temperature (37-42 °C). RPA amplification product can be easily detected by commercial lateral flow strips or 3
real-time fluorescence (Kersting et al., 2014). Recent publications have demonstrated that RPA technology has been successfully applied to the detection of viral, bacterial and parasitic infections (Rohrman and Richards-Kortum, 2012; Boyle et al., 2014; Kersting et al., 2014). In the present study, we established a rapid and simple RPA diagnostic method to test for tropical theileriosis, and evaluated the method using experimentally infected and field-collected blood samples of cattle. The results showed that the RPA assay can be used to detect T. annulata infection using isothermal amplification directly from field blood samples for the first time.
2. Materials and Methods 2.1. Host animals Animal experiments were approved by the Science and Technology Department of Gansu province, China (permit SYXK2010-0003). A 6 to 12-month-old calf was purchased from a Theileria-free area. Total genomic DNA was extracted from the blood of the calf, and DNA samples were detected by PCR using primers specific to the
T.
annulata
cytochrome
b
(5′-CGGTTGGTTTGTTCGTCTTT-3′)
gene and
(cytb),
AnCb-F AnCb-R
(5′-GCCAATGGATTTGAACTTCC-3′) (Junlong et al., 2015). Blood smears were prepared with blood from the ear vein, fixed in methanol for 5 min and stained in 10% Giemsa solution for 30 min (Salih et al., 2007). 2.2. Animal infection and purification of merozoites The calf was infected with T. annulata stabilate cryopreserved in liquid nitrogen from the Vectors and Vector-borne diseases (VVBD) Lab, Lanzhou Veterinary 4
Research Institute (LVRI), China. The animal was monitored daily by measuring the rectal temperature and microscopic examination of Giemsa-stained blood smears post-infection. When the parasitaemia reached 15% on day 25 post-infection, and jugular vein blood was collected in sterile tubes containing citrate anticoagulant for purifying merozoites. T. annulata merozoites were purified using the procedure previously described by Guan et al. (2008) with some modifications. Briefly, infected blood was centrifuged at 1000 × g for 10 min at 4 °C and the packed cells were washed three times with 10 mM Tris-HCl (pH 7.4), 150 mM NaCl (Tris–saline) by centrifugation as described above and then discarded the buffy coat. The residual leukocyte debris was removed by using Transfusion Sets with Leukocyte Reduction Blood Processing Systems (Nanjing, China). The packed erythrocytes were suspended with 7% glycerol solution in Tris-saline at 1:5 (v:v) ratio and then incubated at room temperature for 30 min. After centrifugation as previously described, the supernatant was discarded and the cell pellets were resuspended rapidly with 10 × volume of Tris-saline for lysing erythrocytes at room temperature for 10 min. Lysis was then centrifuged at 1000 × g for 10 min at 4 °C to remove cellular debris and intact erythrocytes. The supernatant was recovered and centrifuged at 4000 × g for 30 min at 4 °C to pellet the merozoites. The pellet of merozoites was washed with Tris–saline and centrifuged until almost free of hemoglobin, and then stored at -70 °C. 2.3. DNA extraction Genomic DNA of T. annulata was extracted from the purified merozoites using 5
the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions and adjusted to a working concentration of 2 ng/μl in TE-buffer (10 mM Tris-HCl 1 mM EDTA, pH 8.0). Seventeen anticoagulated blood samples were collected from tropical theileriosis endemic areas in Gansu province, China. Approximately 10 ml blood samples were collected from the jugular veins of each animal into EDTA vacutainer tubes and were frozen at -20 °C. Blood samples of cattle experimentally infected by T. sinensis, T. sergenti, Babesia bovis, B. bigemina and B. ovata were provided by the VVBD Lab for the specificity of LF-RPA. All of the genomic DNA samples were extracted from 300 μl thawed blood. The DNA concentration was measured using NanoDrop spectrophotometry (Thermo Scientific, USA). DNA samples were stored at -20 °C until use. 2.4. Designing and screening primers and probe A multi-copy gene, cytb of T. annulata (Bilgic et al., 2010), was chosen as the target. Primers and a probe specific to the cytb of T. annulata (GenBank accession no. KP731977) were designed in a conserved region according to the guidelines of TwistDX (Kersting et al., 2014). The specificity and repeatability of several primer pair candidates were tested using the RPA nfo kit (TwistDx, UK), and the amplified products were visualized on a 2% agarose gel to screen for optimal primers. To enable detection by lateral flow strip, the reverse primer was labelled with a biotin at the 5′ end, and the probe was designed to add a 5′ fluorescein FAM, an internal tetrahydrofuran residue (THF) and a C3 spacer (SpC3) on the 3′ end in line with TwistDX. The primers and probe used for RPA reactions were synthesized by Sangon 6
Biotech (Shanghai, China). 2.5. RPA reaction and lateral flow reading RPA reactions were performed using the TwistAmp nfo kit (TwistDx, UK) following the manufacturer′s recommended protocols. Briefly, the amplification mixture of the RPA assay contained 29.5 μl of rehydration buffer, 2.1 μl of each primer (10 μM), 0.6 μl of probe (10 μM), 11.2 μl of ddH2O, and 2 μl of DNA template. A total of 47.5 μl of master mix was aliquoted into each reaction tube supplied with the freeze-dried pellet, and then 2.5 µl of 280 mM magnesium acetate (MgAc) was added to the lid of the tube. The tubes were briefly centrifuged to mix with the MgAc mixed into the solution to initiate the reaction. Samples were then incubated at 37 °C for 20 min under constant shaking at 300 rpm in a Thermo shaker incubator (Thermo, USA). The products were purified with a PCR Purification Kit (Omega, USA) and then examined on a 2% agarose gel. The optimal reaction temperature and time of RPA were explored with various temperature settings (30-50 °C) and different times (5-25 min). The sensitivity of the RPA reaction was determined by using 10-fold serial dilutions of T. annulata genomic DNA ranging from 2 ng/μl to 20 fg/μl, and water was used as a negative control. For the detection of the RPA-generated product directly, Genline Hybridetect−1 lateral flow strips (Milenia Biotec, Germany) were used. After incubation, 2 µl of the amplified product was pipetted and diluted with 98 µl of the running buffer (Milenia Biotec, Germany). Then 10 µl of the sample was transferred to a 1.5 Eppendorf tube containing 200 µl of reaction buffer and the lateral flow strip was placed vertically 7
into the tube for a 5 min incubation at room temperature. A positive result was a clearly visible reddish band at the test line, and a negative control was no band. There was an appearance of a control band to validate the successful test run of the system. 2.6. PCR assay To compare the performance of the lateral flow RPA (LF-RPA) and a PCR assay, a region of the cytb was amplified by PCR using the AnCb-F/AnCb-R (Junlong et al., 2015). The 25 µl PCR reaction contained 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 4 mM MgCl2, 250 μM of each dNTP, 100 pmol of each primer and 1 U Taq polymerase (TaKaRa, China) and was cycled under the conditions previously described by Junlong et al. (2015). All PCR products (393 bp) were analysed on 2% agarose gels stained with ethidium bromide and visualized under ultraviolet light, and the positive products were sequenced and analysed.
3. Results 3.1. Reaction temperature and time optimum of LF-RPA The PCR and microscopic examination showed that the calf was negative for T. annulata, and the high specificity and repeatability of RPA primer pair (designated RPA-F/RPA-R, Table 1) was selected and procuced a 332 bp amplicon. The findings showed that LF-RPA present a positive band over a temperature range of 30-45 °C (Fig. 1A) and in a reaction time of 5 to 25 min using 2 ng genomic DNA. In comparison, a target band could be detected after as little as 5 min incubation on the lateral flow strip, and an optimal amplicon was detected after 10 min at 37 °C (Fig. 1B). 8
3.2. Analytical sensitivity and specificity of LF-RPA The sensitivity of LF-RPA indicated that this assay produced a detectable amplicon at least 2 pg genomic DNA (Fig. 2A). The amplified product achieved a limit of detection of 2 pg genomic DNA as identified on the agarose gel (Fig. 2B). For the specificity of LF-RPA, no cross-detection was observed with the Theileria and Babesia DNA, and only the T. annulata gDNA yielded a positive signal at the test line, which indicated that the primers designed for the LF-RPA reactions were specific to their selected targets (Fig. 3). 3.3. Examination of field samples using the LF-RPA assay A total of 17 field blood samples were simultaneously detected by PCR and LF-RPA assays. In this small number of samples, the LF-RPA assay and PCR results showed a consensus: 10 positive samples were detected by both methods (Additional file 1: Figure S1; Additional file 2: Figure S2).
4. Discussion In the present study, a new method based on RPA in combination with lateral flow was developed to detect T. annulata infection in cattle. The RPA assay targeted the cytb DNA sequence specific to T. annulata, an extrachromosomal DNA with a high copy number and a high level of conservation (Wilson and Williamson, 1997). The cytb of T. annulata is suitable for pathogen detection with higher sensitivity and specificity than other target genes, especially for the carrier cattle in the field (Bilgiç et al., 2010). The RPA technique has been successfully applied to the diagnosis of many 9
parasitic pathogens, such as Cryptosporidium (Crannell et al., 2014), Giardia (Crannell et al., 2015), Leishmania Viannia spp. (Saldarriaga et al., 2016), Plasmodium falciparum (Kersting et al., 2014), Schistosoma haematobium (Rosser et al., 2015) and S. japonicum (Sun et al., 2016). The sensitivity of the LF-RPA assay was found to be equivalent to 0.00002% parasitaemia per reaction, which is similar to the result of loop mediated isothermal amplification (LAMP) in a previous study (Liu et al., 2012). Although LAMP is also an isothermal amplification technique, optimum reaction conditions require amplification temperatures ranging from 60 to 65 °C and a long run time of 30 to 60 min (Notomi et al., 2000). A real-time qPCR assay, which is a simple way to detect T. annulata, has been developed with high sensitivity and specificity, but it is difficult to perform in resource poor areas (Ros-García et al., 2012). Comparatively, the advantages of the LF-RPA assay are impressive: this assay could be performed in less time and could also be easily operated in a water bath or even using body heat in field conditions lacking laboratory equipment (Kersting et al., 2014). Moreover, in addition to its high sensitivity and specificity, the RPA assay is reliable and reproducible in a wide range of temperature (Fig. 1A). Furthermore, the results can be directly observed on the lateral flow strips by the naked eye and easily interpreted by minimally trained people (Rosser et al., 2015). Because RPA offers significant benefits, this method could be as an ideal tool to improve the diagnostic efficiency of T. annulata. In conclusion, the LF-RPA assay is an effective way to detect T. annulata 10
infection with the potential of point-of-care testing. This method can be operated easily and rapidly in resource poor areas. Taken together, this method may contribute to effective surveillance and control of T. annulata disease in endemic areas.
Conflict of interest The authors declare that they have no competing interests.
Acknowledgements This study was financially supported by the 973 Program (No. 2015CB150300); ASTIP, FRIP (No. 2014ZL010), CAAS; NBCIS CARS-38; NSFC (No. 31372432, 31402189); the Science Fund for Youth of Gansu Province (No.145RJYA272); Jiangsu Co-innovation Center programme for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, State Key Laboratory of Veterinary Etiological Biology Project. The research was also facilitated by CRP No. 16198/R0 IAEA.
11
References Ahmed, J., Yin, H., Schnittger, L., Jongejan, F., 2002. Ticks and tick-borne diseases in Asia with special emphasis on China. Parasitol. Res. 88, S51-S55.
Bilgiç, H.B., Karagenç, T., Shiels, B., Tait, A., Eren, H., Weir, W., 2010. Evaluation of cytochrome b as a sensitive target for PCR based detection of T. annulata carrier animals. Vet. Parasitol. 174, 341-347.
Boyle, D.S., McNerney, R., Teng Low, H., Leader, B.T., Pérez-Osorio, A.C., Meyer, J.C., O'Sullivan, D.M., Brooks, D.G., Piepenburg, O., Forrest, M.S., 2014. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification. PLoS One. 9, e103091.
Crannell,
Z.A., Castellanos-Gonzalez,
A., Irani,
A., Rohrman,
B., White,
A.C., Richards-Kortum, R., 2014. Nucleic Acid Test to Diagnose Cryptosporidiosis: Lab Assessment in Animal and Patient Specimens. Anal. Chem. 86, 2565-2571.
Crannell,
Z.A., Cabada,
M.M., Castellanos-Gonzalez,
A., Irani,
A., White,
A.C., Richards-Kortum, R., 2015. Recombinase Polymerase Amplification-Based Assay to Diagnose Giardia in Stool Samples. Am. J. Trop. Med. Hyq. 92, 583-587.
12
Gharbi, M., Sassi, L., Dorchies, P., Darghouth, M.A., 2006. Infection of calves with Theileria annulata in Tunisia: economic analysis and evaluationof the potential benefit of vaccination. Vet. Parasitol. 137, 231-241.
Gomes, J., Inácio, J., 2015. Direct detection of Theileria annulata in bovine blood samples using standard and isothermal DNA amplification approaches. Methods. Mol. Biol. 1247, 175-182.
Guan, G., Chauvin, A., Luo, J., Inoue, N., Moreau, E., Liu, Z., Gao, J., Thekisoe, O.M., Ma, M., Liu, A., Dang, Z., Liu, J., Ren, Q., Jin, Y., Sugimoto, C., Yin, H., 2008. The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China. Exp. Parasitol. 120, 39-44.
Junlong, L., Li, Y., Liu, A., Guan, G., Xie, J., Yin, H., Luo, J., 2015. Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle. Exp. Parasitol. 120, 39-44.
Kersting, S., Rausch, V., Bier, F.F., von Nickisch-Rosenegk, M., 2014. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis. Malar. J. 13, 99. 13
Liu, A., Guan, G., Du, P., Liu, Z., Gou, H., Liu, J., Yang, J., Li, Y., Ma, M., Niu, Q., Ren, Q., Bai, Q., Yin, H., Luo, J., 2012. Loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata infection in China targeting the 18S rRNA and ITS sequences. Exp. Parasitol. 131, 125-129.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T., 2000. Loop-mediated isothermal amplification of DNA. Nucleic. Acids. Res. 28, E63.
Pai, N.P., Vadnais, C., Denkinger, C., Engel, N., Pai, M., 2012. Point-of-Care Testing for Infectious Diseases: Diversity, Complexity, and Barriers in Low- And Middle-Income Countries. PLoS Med. 9, e1001306.
Renneker, S., Kullmann, B., Gerber, S., Dobschanski, J., Bakheit, M.A., Geysen, D., Shiels, B., Tait, A., Ahmed, J.S., Seitzer, U., 2008. Development of a competitive ELISA for detection of Theileria annulata infection. Transbound. Emerg. Dis. 55, 249-256.
Rohrman, B.A., Richards-Kortum, R.R., 2012. A paper and plastic device for performing recombinase polymerase amplification of HIV DNA. Lab. Chip. 12, 3082-3088 14
Ros-García, A., Nicolás, A., García-Pérez, A.L., Juste, R.A., Hurtado, A., 2012. Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle. Parasit. Vectors. 5, 171.
Rosser, A., Rollinson, D., Forrest, M., Webster, B.L., 2015. Isothermal Recombinase Polymerase
amplification
(RPA)
of
Schistosoma
haematobium
DNA and
oligochromatographic lateral flow detection. Parasit. Vectors. 8, 446.
Saldarriaga, O.A., Castellanos-Gonzalez, A., Porrozzi, R., Baldeviano, G.C., Lescano, A.G., de Los Santos, M.B., Fernandez, O.L., Saravia, N.G., Costa, E., Melby, P.C., Travi, B.L., 2016. An Innovative Field-Applicable Molecular Test to Diagnose Cutaneous Leishmania Viannia spp. Infections. PLoS. Negl. Trop. Dis. 10, e0004638.
Salih, D.A., El Hussein, A.M., Seitzer, U., Ahmed, J.S., 2007. Epidemiological studies on tick-borne diseases of cattle in Central Equatoria State, Southern Sudan. Parasitol. Res. 101, 1035-1044.
Sun, K., Xing, W., Yu, X., Fu, W., Wang, Y., Zou, M., Luo, Z., Xu, D., 2016. Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum. Parasit. Vectors. 9, 476.
15
Wilson, R.J., Williamson, D.H., 1997. Extrachromosomal DNA in the Apicomplexa. Microbiol. Mol. Biol. Rev. 61, 1-16.
16
Figure legends Figure 1. The effect of different temperatures and times on the LF-RPA assay. (A) Evaluation of different reaction temperatures from 30-50 °C. (B) Evaluation of different reaction times between 5-25 min. The above line on the lateral flow strip was control line (designated control), the following line was test line (designated test).
17
Figure 2. Sensitivity of RPA assay for the detection of Theileria annulata. Ten-fold serial dilutions of DNA (2 ng/μl to 20 fg/μl) from T. annulata were tested by LF-RPA (A) and by agarose gel electrophoresis (B). NTC: no template controls contained water. M: DL2000TM DNA marker. The above line on the lateral flow strip was control line (designated control), the following line was test line (designated test).
18
Figure 3. Specificity of LF-RPA for different species of piroplasms.
Lane 1: Theileria sinensis; Lane 2: Theileria sergenti; Lane 3: Babesia bovis; Lane 4: Babesia bigemina; Lane 5: Babesia ovata; Lane 6: Positive control; Lane 7: Theileria-free DNA; Lane 8: no template controls contained water.
19
Table1. Recombinase polymerase amplification primers and probe used in the present study Primer name RPA-F RPA-R Lateral flow reverse primer Lateral flow probe
Sequence (5′-3′) 5′-GCTTCTGGGGAGCTACAGTCATAGGTGGTT-3′ 5′-TGCTTGAAAAATAGAAATGAGTCCATAACC-3′ 5′-biotin-TGCTTGAAAAATAGAAATGAGTCCATAACC-3′ 5′-FAM-GGAGGCCAAACAGTTGGTCCAGAGACATTA(THF)- AGAGATTCTTTTCTATAC-SpC3-3′
20
S0304-4017(17)30070-5 http://dx.doi.org/doi:10.1016/j.vetpar.2017.02.019 VETPAR 8268
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
23-11-2016 14-2-2017 18-2-2017
Please cite this article as: Yin, Fangyuan, Liu, Junlong, Liu, Aihong, Li, Youquan, Luo, Jianxun, Guan, Guiquan, Yin, Hong, Rapid diagnosis of Theileria annulata by recombinase polymerase amplification combined with a lateral flow strip (LF-RPA) in epidemic regions.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2017.02.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Rapid diagnosis of Theileria annulata by recombinase polymerase amplification combined with a lateral flow strip (LF-RPA) in epidemic regions
Fangyuan Yin a, Junlong Liu a, Aihong Liu a, Youquan Li a, Jianxun Luo a, Guiquan Guan a*, Hong Yin a, b*
a
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of
Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Gansu 730046, P. R. China. b
Jiangsu Co-innovation Center for Prevention and Control of Important Animal
Infectious Diseases and Zoonoses, Yangzhou, 225009, P. R. China.
*Corresponding authors: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu 730046, P. R. China. E-mail: [email protected]; [email protected] Tel.: +86 931 8342681 Fax: +86 931 8340977
1
Highlights
A LF-RPA was firstly developed to detect Theileria annulata infections.
The sensitivity of LF-RPA is similar to PCR for detecting the field samples.
The LF-RPA could be used to surveil and control of Theileria annulata.
Abstract Rapid and accurate diagnosis of Theileria annulata infection contributes to the formulation of strategies to eradicate this parasite. A simple and efficient diagnostic tool, recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip, was used in detection of Theileria and compared to other methods that require expensive instruments and skilled personnel. Herein, we established and optimized an LF-RPA method to detect the cytochrome b gene of T. annulata mitochondrial DNA from experimentally infected and field-collected blood samples. This method has many unparalleled characteristics, including that it is rapid (clear detection in 5 min at constant temperature), sensitive (the limitation of detection is at least 2 pg genomic DNA), and specific (no cross-reaction with other piroplasms that infect cattle). The LF-RPA assay was evaluated via testing 17 field blood samples and comparing the results of that of a PCR, showing 100% agreement, which demonstrates the ability of the LF-RPA assay to detect T. annulata infections in small number of samples (n = 17). Taken together, the results indicate that this method could be used as an ideal 2
diagnostic tool for detecting T. annulata in endemic regions with limited to fewer and local resources and could also be a potential technique for the surveillance and control of blood protozoa.
Keywords: Theileria annulata; Diagnosis; Recombinase polymerase amplification; Lateral flow;
1. Introduction Theileria annulata, one of the tick-borne protozoan parasites infective to ruminants, is the pathogen of tropical theileriosis and causes great losses to the cattle industry worldwide. (Gharbi et al., 2006). Currently, diagnostic tests mainly consist of microscopic examinations (Ahmed et al., 2002), serological assays (Renneker et al., 2008) and PCR-based detection (Gomes and Inácio, 2015) for effective prevention and control of tropical theileriosis. However, these methods are not well suited for direct field testing because of their complicated operation processes, trained personnel and time consuming (Pai et al., 2012). It is essential to develop a rapid, simple and clinical applications test to improve the efficacy of current control programmes. Recombinase polymerase amplification is a novel isothermal amplification method that does not require sophisticated equipment or thermal denaturation and can be performed for less than 20 min at a low and constant temperature (37-42 °C). RPA amplification product can be easily detected by commercial lateral flow strips or 3
real-time fluorescence (Kersting et al., 2014). Recent publications have demonstrated that RPA technology has been successfully applied to the detection of viral, bacterial and parasitic infections (Rohrman and Richards-Kortum, 2012; Boyle et al., 2014; Kersting et al., 2014). In the present study, we established a rapid and simple RPA diagnostic method to test for tropical theileriosis, and evaluated the method using experimentally infected and field-collected blood samples of cattle. The results showed that the RPA assay can be used to detect T. annulata infection using isothermal amplification directly from field blood samples for the first time.
2. Materials and Methods 2.1. Host animals Animal experiments were approved by the Science and Technology Department of Gansu province, China (permit SYXK2010-0003). A 6 to 12-month-old calf was purchased from a Theileria-free area. Total genomic DNA was extracted from the blood of the calf, and DNA samples were detected by PCR using primers specific to the
T.
annulata
cytochrome
b
(5′-CGGTTGGTTTGTTCGTCTTT-3′)
gene and
(cytb),
AnCb-F AnCb-R
(5′-GCCAATGGATTTGAACTTCC-3′) (Junlong et al., 2015). Blood smears were prepared with blood from the ear vein, fixed in methanol for 5 min and stained in 10% Giemsa solution for 30 min (Salih et al., 2007). 2.2. Animal infection and purification of merozoites The calf was infected with T. annulata stabilate cryopreserved in liquid nitrogen from the Vectors and Vector-borne diseases (VVBD) Lab, Lanzhou Veterinary 4
Research Institute (LVRI), China. The animal was monitored daily by measuring the rectal temperature and microscopic examination of Giemsa-stained blood smears post-infection. When the parasitaemia reached 15% on day 25 post-infection, and jugular vein blood was collected in sterile tubes containing citrate anticoagulant for purifying merozoites. T. annulata merozoites were purified using the procedure previously described by Guan et al. (2008) with some modifications. Briefly, infected blood was centrifuged at 1000 × g for 10 min at 4 °C and the packed cells were washed three times with 10 mM Tris-HCl (pH 7.4), 150 mM NaCl (Tris–saline) by centrifugation as described above and then discarded the buffy coat. The residual leukocyte debris was removed by using Transfusion Sets with Leukocyte Reduction Blood Processing Systems (Nanjing, China). The packed erythrocytes were suspended with 7% glycerol solution in Tris-saline at 1:5 (v:v) ratio and then incubated at room temperature for 30 min. After centrifugation as previously described, the supernatant was discarded and the cell pellets were resuspended rapidly with 10 × volume of Tris-saline for lysing erythrocytes at room temperature for 10 min. Lysis was then centrifuged at 1000 × g for 10 min at 4 °C to remove cellular debris and intact erythrocytes. The supernatant was recovered and centrifuged at 4000 × g for 30 min at 4 °C to pellet the merozoites. The pellet of merozoites was washed with Tris–saline and centrifuged until almost free of hemoglobin, and then stored at -70 °C. 2.3. DNA extraction Genomic DNA of T. annulata was extracted from the purified merozoites using 5
the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions and adjusted to a working concentration of 2 ng/μl in TE-buffer (10 mM Tris-HCl 1 mM EDTA, pH 8.0). Seventeen anticoagulated blood samples were collected from tropical theileriosis endemic areas in Gansu province, China. Approximately 10 ml blood samples were collected from the jugular veins of each animal into EDTA vacutainer tubes and were frozen at -20 °C. Blood samples of cattle experimentally infected by T. sinensis, T. sergenti, Babesia bovis, B. bigemina and B. ovata were provided by the VVBD Lab for the specificity of LF-RPA. All of the genomic DNA samples were extracted from 300 μl thawed blood. The DNA concentration was measured using NanoDrop spectrophotometry (Thermo Scientific, USA). DNA samples were stored at -20 °C until use. 2.4. Designing and screening primers and probe A multi-copy gene, cytb of T. annulata (Bilgic et al., 2010), was chosen as the target. Primers and a probe specific to the cytb of T. annulata (GenBank accession no. KP731977) were designed in a conserved region according to the guidelines of TwistDX (Kersting et al., 2014). The specificity and repeatability of several primer pair candidates were tested using the RPA nfo kit (TwistDx, UK), and the amplified products were visualized on a 2% agarose gel to screen for optimal primers. To enable detection by lateral flow strip, the reverse primer was labelled with a biotin at the 5′ end, and the probe was designed to add a 5′ fluorescein FAM, an internal tetrahydrofuran residue (THF) and a C3 spacer (SpC3) on the 3′ end in line with TwistDX. The primers and probe used for RPA reactions were synthesized by Sangon 6
Biotech (Shanghai, China). 2.5. RPA reaction and lateral flow reading RPA reactions were performed using the TwistAmp nfo kit (TwistDx, UK) following the manufacturer′s recommended protocols. Briefly, the amplification mixture of the RPA assay contained 29.5 μl of rehydration buffer, 2.1 μl of each primer (10 μM), 0.6 μl of probe (10 μM), 11.2 μl of ddH2O, and 2 μl of DNA template. A total of 47.5 μl of master mix was aliquoted into each reaction tube supplied with the freeze-dried pellet, and then 2.5 µl of 280 mM magnesium acetate (MgAc) was added to the lid of the tube. The tubes were briefly centrifuged to mix with the MgAc mixed into the solution to initiate the reaction. Samples were then incubated at 37 °C for 20 min under constant shaking at 300 rpm in a Thermo shaker incubator (Thermo, USA). The products were purified with a PCR Purification Kit (Omega, USA) and then examined on a 2% agarose gel. The optimal reaction temperature and time of RPA were explored with various temperature settings (30-50 °C) and different times (5-25 min). The sensitivity of the RPA reaction was determined by using 10-fold serial dilutions of T. annulata genomic DNA ranging from 2 ng/μl to 20 fg/μl, and water was used as a negative control. For the detection of the RPA-generated product directly, Genline Hybridetect−1 lateral flow strips (Milenia Biotec, Germany) were used. After incubation, 2 µl of the amplified product was pipetted and diluted with 98 µl of the running buffer (Milenia Biotec, Germany). Then 10 µl of the sample was transferred to a 1.5 Eppendorf tube containing 200 µl of reaction buffer and the lateral flow strip was placed vertically 7
into the tube for a 5 min incubation at room temperature. A positive result was a clearly visible reddish band at the test line, and a negative control was no band. There was an appearance of a control band to validate the successful test run of the system. 2.6. PCR assay To compare the performance of the lateral flow RPA (LF-RPA) and a PCR assay, a region of the cytb was amplified by PCR using the AnCb-F/AnCb-R (Junlong et al., 2015). The 25 µl PCR reaction contained 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 4 mM MgCl2, 250 μM of each dNTP, 100 pmol of each primer and 1 U Taq polymerase (TaKaRa, China) and was cycled under the conditions previously described by Junlong et al. (2015). All PCR products (393 bp) were analysed on 2% agarose gels stained with ethidium bromide and visualized under ultraviolet light, and the positive products were sequenced and analysed.
3. Results 3.1. Reaction temperature and time optimum of LF-RPA The PCR and microscopic examination showed that the calf was negative for T. annulata, and the high specificity and repeatability of RPA primer pair (designated RPA-F/RPA-R, Table 1) was selected and procuced a 332 bp amplicon. The findings showed that LF-RPA present a positive band over a temperature range of 30-45 °C (Fig. 1A) and in a reaction time of 5 to 25 min using 2 ng genomic DNA. In comparison, a target band could be detected after as little as 5 min incubation on the lateral flow strip, and an optimal amplicon was detected after 10 min at 37 °C (Fig. 1B). 8
3.2. Analytical sensitivity and specificity of LF-RPA The sensitivity of LF-RPA indicated that this assay produced a detectable amplicon at least 2 pg genomic DNA (Fig. 2A). The amplified product achieved a limit of detection of 2 pg genomic DNA as identified on the agarose gel (Fig. 2B). For the specificity of LF-RPA, no cross-detection was observed with the Theileria and Babesia DNA, and only the T. annulata gDNA yielded a positive signal at the test line, which indicated that the primers designed for the LF-RPA reactions were specific to their selected targets (Fig. 3). 3.3. Examination of field samples using the LF-RPA assay A total of 17 field blood samples were simultaneously detected by PCR and LF-RPA assays. In this small number of samples, the LF-RPA assay and PCR results showed a consensus: 10 positive samples were detected by both methods (Additional file 1: Figure S1; Additional file 2: Figure S2).
4. Discussion In the present study, a new method based on RPA in combination with lateral flow was developed to detect T. annulata infection in cattle. The RPA assay targeted the cytb DNA sequence specific to T. annulata, an extrachromosomal DNA with a high copy number and a high level of conservation (Wilson and Williamson, 1997). The cytb of T. annulata is suitable for pathogen detection with higher sensitivity and specificity than other target genes, especially for the carrier cattle in the field (Bilgiç et al., 2010). The RPA technique has been successfully applied to the diagnosis of many 9
parasitic pathogens, such as Cryptosporidium (Crannell et al., 2014), Giardia (Crannell et al., 2015), Leishmania Viannia spp. (Saldarriaga et al., 2016), Plasmodium falciparum (Kersting et al., 2014), Schistosoma haematobium (Rosser et al., 2015) and S. japonicum (Sun et al., 2016). The sensitivity of the LF-RPA assay was found to be equivalent to 0.00002% parasitaemia per reaction, which is similar to the result of loop mediated isothermal amplification (LAMP) in a previous study (Liu et al., 2012). Although LAMP is also an isothermal amplification technique, optimum reaction conditions require amplification temperatures ranging from 60 to 65 °C and a long run time of 30 to 60 min (Notomi et al., 2000). A real-time qPCR assay, which is a simple way to detect T. annulata, has been developed with high sensitivity and specificity, but it is difficult to perform in resource poor areas (Ros-García et al., 2012). Comparatively, the advantages of the LF-RPA assay are impressive: this assay could be performed in less time and could also be easily operated in a water bath or even using body heat in field conditions lacking laboratory equipment (Kersting et al., 2014). Moreover, in addition to its high sensitivity and specificity, the RPA assay is reliable and reproducible in a wide range of temperature (Fig. 1A). Furthermore, the results can be directly observed on the lateral flow strips by the naked eye and easily interpreted by minimally trained people (Rosser et al., 2015). Because RPA offers significant benefits, this method could be as an ideal tool to improve the diagnostic efficiency of T. annulata. In conclusion, the LF-RPA assay is an effective way to detect T. annulata 10
infection with the potential of point-of-care testing. This method can be operated easily and rapidly in resource poor areas. Taken together, this method may contribute to effective surveillance and control of T. annulata disease in endemic areas.
Conflict of interest The authors declare that they have no competing interests.
Acknowledgements This study was financially supported by the 973 Program (No. 2015CB150300); ASTIP, FRIP (No. 2014ZL010), CAAS; NBCIS CARS-38; NSFC (No. 31372432, 31402189); the Science Fund for Youth of Gansu Province (No.145RJYA272); Jiangsu Co-innovation Center programme for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, State Key Laboratory of Veterinary Etiological Biology Project. The research was also facilitated by CRP No. 16198/R0 IAEA.
11
References Ahmed, J., Yin, H., Schnittger, L., Jongejan, F., 2002. Ticks and tick-borne diseases in Asia with special emphasis on China. Parasitol. Res. 88, S51-S55.
Bilgiç, H.B., Karagenç, T., Shiels, B., Tait, A., Eren, H., Weir, W., 2010. Evaluation of cytochrome b as a sensitive target for PCR based detection of T. annulata carrier animals. Vet. Parasitol. 174, 341-347.
Boyle, D.S., McNerney, R., Teng Low, H., Leader, B.T., Pérez-Osorio, A.C., Meyer, J.C., O'Sullivan, D.M., Brooks, D.G., Piepenburg, O., Forrest, M.S., 2014. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification. PLoS One. 9, e103091.
Crannell,
Z.A., Castellanos-Gonzalez,
A., Irani,
A., Rohrman,
B., White,
A.C., Richards-Kortum, R., 2014. Nucleic Acid Test to Diagnose Cryptosporidiosis: Lab Assessment in Animal and Patient Specimens. Anal. Chem. 86, 2565-2571.
Crannell,
Z.A., Cabada,
M.M., Castellanos-Gonzalez,
A., Irani,
A., White,
A.C., Richards-Kortum, R., 2015. Recombinase Polymerase Amplification-Based Assay to Diagnose Giardia in Stool Samples. Am. J. Trop. Med. Hyq. 92, 583-587.
12
Gharbi, M., Sassi, L., Dorchies, P., Darghouth, M.A., 2006. Infection of calves with Theileria annulata in Tunisia: economic analysis and evaluationof the potential benefit of vaccination. Vet. Parasitol. 137, 231-241.
Gomes, J., Inácio, J., 2015. Direct detection of Theileria annulata in bovine blood samples using standard and isothermal DNA amplification approaches. Methods. Mol. Biol. 1247, 175-182.
Guan, G., Chauvin, A., Luo, J., Inoue, N., Moreau, E., Liu, Z., Gao, J., Thekisoe, O.M., Ma, M., Liu, A., Dang, Z., Liu, J., Ren, Q., Jin, Y., Sugimoto, C., Yin, H., 2008. The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China. Exp. Parasitol. 120, 39-44.
Junlong, L., Li, Y., Liu, A., Guan, G., Xie, J., Yin, H., Luo, J., 2015. Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle. Exp. Parasitol. 120, 39-44.
Kersting, S., Rausch, V., Bier, F.F., von Nickisch-Rosenegk, M., 2014. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis. Malar. J. 13, 99. 13
Liu, A., Guan, G., Du, P., Liu, Z., Gou, H., Liu, J., Yang, J., Li, Y., Ma, M., Niu, Q., Ren, Q., Bai, Q., Yin, H., Luo, J., 2012. Loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata infection in China targeting the 18S rRNA and ITS sequences. Exp. Parasitol. 131, 125-129.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T., 2000. Loop-mediated isothermal amplification of DNA. Nucleic. Acids. Res. 28, E63.
Pai, N.P., Vadnais, C., Denkinger, C., Engel, N., Pai, M., 2012. Point-of-Care Testing for Infectious Diseases: Diversity, Complexity, and Barriers in Low- And Middle-Income Countries. PLoS Med. 9, e1001306.
Renneker, S., Kullmann, B., Gerber, S., Dobschanski, J., Bakheit, M.A., Geysen, D., Shiels, B., Tait, A., Ahmed, J.S., Seitzer, U., 2008. Development of a competitive ELISA for detection of Theileria annulata infection. Transbound. Emerg. Dis. 55, 249-256.
Rohrman, B.A., Richards-Kortum, R.R., 2012. A paper and plastic device for performing recombinase polymerase amplification of HIV DNA. Lab. Chip. 12, 3082-3088 14
Ros-García, A., Nicolás, A., García-Pérez, A.L., Juste, R.A., Hurtado, A., 2012. Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle. Parasit. Vectors. 5, 171.
Rosser, A., Rollinson, D., Forrest, M., Webster, B.L., 2015. Isothermal Recombinase Polymerase
amplification
(RPA)
of
Schistosoma
haematobium
DNA and
oligochromatographic lateral flow detection. Parasit. Vectors. 8, 446.
Saldarriaga, O.A., Castellanos-Gonzalez, A., Porrozzi, R., Baldeviano, G.C., Lescano, A.G., de Los Santos, M.B., Fernandez, O.L., Saravia, N.G., Costa, E., Melby, P.C., Travi, B.L., 2016. An Innovative Field-Applicable Molecular Test to Diagnose Cutaneous Leishmania Viannia spp. Infections. PLoS. Negl. Trop. Dis. 10, e0004638.
Salih, D.A., El Hussein, A.M., Seitzer, U., Ahmed, J.S., 2007. Epidemiological studies on tick-borne diseases of cattle in Central Equatoria State, Southern Sudan. Parasitol. Res. 101, 1035-1044.
Sun, K., Xing, W., Yu, X., Fu, W., Wang, Y., Zou, M., Luo, Z., Xu, D., 2016. Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum. Parasit. Vectors. 9, 476.
15
Wilson, R.J., Williamson, D.H., 1997. Extrachromosomal DNA in the Apicomplexa. Microbiol. Mol. Biol. Rev. 61, 1-16.
16
Figure legends Figure 1. The effect of different temperatures and times on the LF-RPA assay. (A) Evaluation of different reaction temperatures from 30-50 °C. (B) Evaluation of different reaction times between 5-25 min. The above line on the lateral flow strip was control line (designated control), the following line was test line (designated test).
17
Figure 2. Sensitivity of RPA assay for the detection of Theileria annulata. Ten-fold serial dilutions of DNA (2 ng/μl to 20 fg/μl) from T. annulata were tested by LF-RPA (A) and by agarose gel electrophoresis (B). NTC: no template controls contained water. M: DL2000TM DNA marker. The above line on the lateral flow strip was control line (designated control), the following line was test line (designated test).
18
Figure 3. Specificity of LF-RPA for different species of piroplasms.
Lane 1: Theileria sinensis; Lane 2: Theileria sergenti; Lane 3: Babesia bovis; Lane 4: Babesia bigemina; Lane 5: Babesia ovata; Lane 6: Positive control; Lane 7: Theileria-free DNA; Lane 8: no template controls contained water.
19
Table1. Recombinase polymerase amplification primers and probe used in the present study Primer name RPA-F RPA-R Lateral flow reverse primer Lateral flow probe
Sequence (5′-3′) 5′-GCTTCTGGGGAGCTACAGTCATAGGTGGTT-3′ 5′-TGCTTGAAAAATAGAAATGAGTCCATAACC-3′ 5′-biotin-TGCTTGAAAAATAGAAATGAGTCCATAACC-3′ 5′-FAM-GGAGGCCAAACAGTTGGTCCAGAGACATTA(THF)- AGAGATTCTTTTCTATAC-SpC3-3′
20