Patent Publication Number: US-2022235399-A1

Title: Use of p44 as marker for diagnosing anaplasmosis

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is the U.S. national stage of PCT/KR2020/005252 filed on Apr. 21, 2020, which claims priority of Korean Patent Application No. 10-2019-0049225 filed on April 26, 2019, the contents of which are incorporated herein. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 26, 2021, is named 272517_500980_ST25.txt and is 11,747 bytes in size. 
     TECHNICAL FIELD 
     The present disclosure relates to a novel use of P 44  as a marker for predicting or diagnosing anaplasmosis. 
     BACKGROUND 
     Anaplasmosis is an acute febrile illness that is transmitted through the bite of a tick carrying  Anaplasma phagocytophilum  and exhibits a nonspecific acute febrile clinical pattern. Young adults with anaplasmosis exhibit mild symptoms, whereas elderly or immunocompromised anaplasmosis patients exhibit thrombocytopenia, leukopenia, elevated levels of gamma glutamyltransferase, and other more severe symptoms. Anaplasmosis responds well to antibiotic treatment, but some patients may die due to complications if diagnosis is delayed. Therefore, prompt and accurate diagnosis is essential for ameliorating the prognosis of patients. 
     Currently used methods of diagnosing anaplasmosis include detecting anaplasmosis by isolating  A. phagocytophilum  from the blood of a patient and culturing the same, detecting morulae at an early stage in peripheral blood samples through Wright-Giemsa staining using peripheral blood, performing detection in patient sera using an antibody to  A. phagocytophilum , and the like. 
     However, methods of diagnosing disease by culturing  A. phagocytophilum  require long culture time of several weeks or more, and thus are unsuitable for clinical diagnosis in practice. Indirect immunofluorescence antibody and immunoenzyme methods, which are serological tests, have disadvantages of causing false-positive reactions upon cross-reactivity with other pathogens, exhibiting low sensitivity when used for early disease detection because it takes several days for antibodies to form after the onset of symptoms, and requiring follow-up examination for definitive diagnosis. 
     In addition, when a very low cut-off values of IgM 1:16 or higher and IgG 1:80 or higher are used as diagnostic criteria for anaplasmosis in a clinical study targeting anaplasmosis patients, IgM sensitivity is only 23% and IgG sensitivity is only 15%, indicating that the current diagnostic method has problems of low speed and low accuracy. 
     Furthermore, in order to diagnose anaplasmosis using PCR, the 16S rRNA, ankA, and groEL target protein genes were used as diagnostic biomarkers. When these genes are employed as diagnostic markers, it is inconvenient since nested PCR and real-time PCR should be used instead of conventional PCR. Because  A. phagocytophilum  is an intracellular bacterium, the detection sensitivity of conventional PCR is low. Although conducting nested PCR twice improve the sensitivity, this is cumbersome because it consumes more labor and time than necessary and entails increased potential for contamination. Therefore, it is critical to develop a novel diagnostic method that is capable of exhibiting excellent sensitivity without being performed repeatedly. 
     Accordingly, the present inventors identified P 44 , which is a multi-copy gene that can be used as a novel biomarker for quick and accurate diagnosis of anaplasmosis with high sensitivity, and devised a primer set capable of detecting anaplasmosis and a kit including the same, thereby completing the present disclosure. 
     SUMMARY 
     Therefore, the present disclosure has been made in view of the above problems, and it is one object of the present disclosure to provide a composition for anaplasmosis diagnostic markers containing a P 44  gene. 
     Other objects of the present disclosure include providing a diagnostic composition for anaplasmosis containing a substance for measuring the level of the P 44  gene or the P 44  protein. A primer set or probe designed to identify  Anaplasma phagocytophilum . A kit for diagnosing anaplasmosis containing the present disclosure&#39;s diagnostic composition for anaplasmosis and a method for providing information to diagnose  Anaplasma phagocytophilum  infection using the present disclosure&#39;s diagnostic kit for anaplasmosis. 
     In accordance with the present disclosure, the above and other objects can be accomplished by the provision of a composition as a diagnostic marker for anaplasmosis containing a P 44  gene. 
     In one embodiment of the present disclosure, the P 44  gene may have the nucleotide sequence of SEQ ID NO: 1. 
     In accordance with another aspect of the present disclosure, provided is a diagnostic composition for anaplasmosis containing a substance for measuring the level of a P 44  gene or P 44  protein. 
     In one embodiment of the present disclosure, the substance for measuring the level of the P 44  gene may be a primer or probe that specifically binds to the P 44  gene or P 44  mRNA. 
     In one embodiment of the present disclosure, the primer may be a primer set selected from the group consisting of a primer set consisting of primers having sequences of SEQ ID NOS: 3 and 4, a primer set consisting of primers having sequences of SEQ ID NOS: 5 and 6, a primer set consisting of primers having sequences of SEQ ID NOS: 7 and 8, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 10, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 11, a primer set consisting of primers having sequences of SEQ ID NOS: 12 and 13, and a primer set consisting of primers having sequences of SEQ ID NOS: 14 and 15, and the probe may be selected from the group consisting of probes having sequences of SEQ ID NOS: 16 to 27. 
     In one embodiment of the present disclosure, the substance for measuring the level of the P 44  protein may be an antibody that specifically recognizes the P 44  protein. 
     In accordance with another aspect of the present disclosure, provided is a primer set for detecting  Anaplasma phagocytophilum , wherein the primer set is selected from the group consisting of: a primer set consisting of primers having sequences of SEQ ID NOS: 3 and 4, a primer set consisting of primers having sequences of SEQ ID NOS: 5 and 6, a primer set consisting of primers having sequences of SEQ ID NOS: 7 and 8, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 10, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 11, a primer set consisting of primers having sequences of SEQ ID NOS: 12 and 13, and a primer set consisting of primers having sequences of SEQ ID NOS: 14 and 15. 
     In accordance with another aspect of the present disclosure, provided is a probe for detecting  Anaplasma phagocytophilum , wherein the probe is selected from the group consisting of probes having sequences of SEQ ID NOS: 16 to 27. 
     In accordance with another aspect of the present disclosure, provided is a diagnostic kit for anaplasmosis containing the diagnostic composition for anaplasmosis of the present disclosure. 
     In one embodiment of the present disclosure, the diagnostic kit may include at least one selected from the group consisting of: a primer set consisting of primers having sequences of SEQ ID NOS: 3 and 4, a primer set consisting of primers having sequences of SEQ ID NOS: 5 and 6, a primer set consisting of primers having sequences of SEQ ID NOS: 7 and 8, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 10, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 11, a primer set consisting of primers having sequences of SEQ ID NOS: 12 and 13, a primer set consisting of primers having sequences of SEQ ID NOS: 14 and 15, and a probe selected from the group consisting of probes having sequences of SEQ ID NOS: 16 to 27. 
     In accordance with another aspect of the present disclosure, provided is a method for providing information to diagnose infection with anaplasmosis using the diagnostic kit for anaplasmosis containing the diagnostic composition of the present disclosure. 
     In one embodiment of the present disclosure, the diagnostic kit for anaplasmosis may perform a polymerase chain reaction (PCR) using any one primer set selected from the group consisting of: a primer set consisting of primers having sequences of SEQ ID NOS: 3 and 4, a primer set consisting of primers having sequences of SEQ ID NOS: 5 and 6, a primer set consisting of primers having sequences of SEQ ID NOS: 7 and 8, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 10, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 11, a primer set consisting of primers having sequences of SEQ ID NOS: 12 and 13, and a primer set consisting of primers having sequences of SEQ ID NOS: 14 and 15, or any one probe selected from the group consisting of probes having sequences of SEQ ID NOS: 16 to 27. 
     In one embodiment of the present disclosure, the diagnosis is performed using a PCR method selected from the group consisting of conventional polymerase chain reaction (C-PCR: conventional PCR), nested polymerase chain reaction (N-PCR: nested PCR), multiple polymerase chain reaction, real-time polymerase chain reaction, real-time quantitative polymerase chain reaction, and loop-mediated isothermal amplification (LAMP). 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The aforementioned and other objects, features and other advantages of the present disclosure will provide a clearly understanding from the following detailed description taken in conjunction with the accompanying drawings, in which: 
         FIG. 1  illustrates the result of a comparative analysis (ROC curve) on the sensitivity of anaplasmosis diagnosis using various PCR methods employing P44, a novel marker for diagnosing anaplasmosis according to the present disclosure, and conventional markers. 
     
    
    
     DETAILED DESCRIPTION 
     The present disclosure is characterized by identifying the fact that P 44  can be used as a novel biomarker that can accurately and quickly diagnose anaplasmosis at an early stage. 
     In particular, when searching a method to enhance the speed and accuracy of anaplasmosis diagnosis, the present inventors identified that P 44 , a multi-copy gene present in  A. phagocytophilum , can be used as a novel biomarker for diagnosing anaplasmosis. 
     In particular, the novel biomarker targeted by the present disclosure is a multi-copy gene. The reason for this is that the multi-copy gene exists as a repeating sequence in a gene cluster, allowing multiple copies to exist in one cell, resulting in extremely high sensitivity. Therefore, a biomarker capable of detecting  A. phagocytophilum  was screened from multi-copy genes. 
     It was found that, among them, P 44  is capable of specifically detecting  A. phagocytophilum  with higher sensitivity than other polyclonal genes when used as a marker. 
     Accordingly, the present disclosure provides a composition as a diagnostic marker for anaplasmosis containing a P 44  gene. 
     Anaplasmosis (human granulocytic anaplasmosis, HGA) is also known as zoonosis, and infects humans, dogs, cattle, sheep, goats, horses and wild animals, and the causative pathogen thereof is a bacterium called “ Anaplasma phagocytophilum ” that is obligately parasitic within cells. 
     Therefore, “anaplasmosis” in the present disclosure refers to a disease caused by infection with  Anaplasma phagocytophilum.    
     As used herein, the term “diagnosis” refers to determine a subject&#39;s susceptibility to a certain disease or disorder, determine whether or not a subject currently has a specific disease or disorder, or determine the prognosis of a subject with a specific disease or disorder, or therametrics (e.g., monitoring the condition of a subject to provide information about therapeutic efficacy). 
     As used herein, the term “marker” refers to a substance that can be used to determine whether or not a subject is infected with  Anaplasma phagocytophilum , or a substance that can be used to distinguish a subject with anaplasmosis caused by infection with  Anaplasma phagocytophilum  from a normal subject, and includes organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, and the like). For the purposes of the present disclosure, the diagnostic marker for anaplasmosis may be a P44 gene or a protein expressed by the gene. 
     Preferably, the P 44  gene of the present disclosure may include the nucleotide sequence of SEQ ID NO: 1, and the P 44  protein expressed by the gene may include the amino acid sequence of SEQ ID NO: 2. 
     In addition, the present disclosure provides a diagnostic composition for anaplasmosis containing a substance for measuring the level of the P 44  gene or P 44  protein. 
     That is, the diagnostic composition can detect  Anaplasma phagocytophilum , the causative pathogen of anaplasmosis, and the detection may be performed using polymerase chain reaction (PCR). 
     The substance for measuring the level of the P 44  gene may be a primer or probe that specifically binds to the P 44  gene or P 44  mRNA, and preferably, the primer is selected from the group consisting of: a primer set consisting of primers having sequences of SEQ ID NOS: 3 and 4, a primer set consisting of primers having sequences of SEQ ID NOS: 5 and 6, a primer set consisting of primers having sequences of SEQ ID NOS: 7 and 8, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 10, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 11, a primer set consisting of primers having sequences of SEQ ID NOS: 12 and 13, and a primer set consisting of primers having sequences of SEQ ID NOS: 14 and 15, and the probe may be selected from the group consisting of probes having sequences of SEQ ID NOS: 16 to 27. 
     In addition, the substance for measuring the level of the P 44  protein may be an antibody that specifically recognizes the P 44  protein. 
     The diagnostic composition of the present disclosure may contain at least one component, for example, a buffer solution for reaction, dNTP, Mg 2+  ions, and DNA polymerase, that may be used for polymerase chain reaction (PCR), in addition to the respective primer sets or probe described above. 
     The buffer solution for reaction may be 1 to 10 mM Tris HCl or 10 to 40 mM KCl (pH 9.0), and the dNTP may comprise at least one of dATP, dTTP, dGTP, and dCTP. 
     The diagnostic composition may include a stabilizer and/or a non-reactive dye to improve experimental convenience, stability, and reactivity. 
     The non-reactive dye should be selected from substances that do not affect the polymerase chain reaction, and is intended to be used for analysis or identification using the polymerase chain reaction product. Substances that satisfy these requirements may be water-soluble dyes such as rhodamine, TAMRA, sodium hypochlorite (household lax), bromophenol blue, xylene cyanol, bromocresol red, and cresol red. 
     The diagnostic composition may be provided in a liquid form, and is preferably provided in a dried state to increase stability, convenience of storage, and long-term storage. The drying may be performed by a known drying method such as general room-temperature drying, heat drying, freeze drying, or vacuum drying, but any drying method may be used as long as it does not cause loss of components of the composition. The drying method described above may vary depending on the type and amount of the enzyme that is used. 
     The diagnostic composition is produced and utilized in a stable manner by mixing in a single reaction tube, followed by freezing or drying, thus requiring no separate mixing steps during the polymerase chain reaction. Therefore, errors due to mixing during the reaction can be prevented and stability, reactivity, and storability can be improved. 
     A commercially available product that contains ingredients other than the primer set or probe, namely, buffer solution for reaction, dNTP, Mg 2+  ion, and DNA polymerase, may be utilized as the diagnostic composition. 
     The present disclosure further includes a primer set or probe that is capable of specifically detecting  Anaplasma phagocytophilum.    
     The primer or probe according to the present disclosure may be a primer or probe that specifically binds to P 44  identified in the present disclosure in order to rapidly and accurately detect  Anaplasma phagocytophilum , and the primer is preferably selected from the group consisting of: a primer set consisting of primers having sequences of SEQ ID NOS: 3 and 4, a primer set consisting of primers having sequences of SEQ ID 
     NOS: 5 and 6, a primer set consisting of primers having sequences of SEQ ID NOS: 7 and 8, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 10, a primer set consisting of primers having sequences of SEQ ID NOS: 9 and 11, a primer set consisting of primers having sequences of SEQ ID NOS: 12 and 13, and a primer set consisting of primers having sequences of SEQ ID NOS: 14 and 15. 
     In addition, preferably, the probe may be selected from the group including SEQ ID NOS: 16 to 27. 
     In particular, the primer or probe designed in the present disclosure aims to enable rapid and accurate detection of  Anaplasma phagocytophilum  at an early stage, and can specifically bind to and/or amplify the multi-copy gene P 44 , and is useful for polymerase chain reaction (PCR). 
     Furthermore, the present disclosure provides a diagnostic kit for detecting  Anaplasma phagocytophilum.    
     The diagnostic kit contains the diagnostic composition for anaplasmosis of the present disclosure as described above. 
     Using the diagnostic kit, the present disclosure also provides a method of providing information for diagnosing whether or not a subject is infected with  Anaplasma phagocytophilum    
     Specifically, the present disclosure provides a method of providing information to diagnose infection with anaplasmosis, the method including mixing a DNA-containing sample isolated from a biological sample obtained from a subject suspected of having contracted anaplasmosis with the diagnostic composition of the present disclosure as described above, performing a reaction to amplify the reaction mixture, and analyzing the resultant amplification product. 
     The diagnostic composition includes the above-described primer set or probe prepared in the present disclosure, and the reaction to amplify the reaction mixture may be performed by a polymerase chain reaction (PCR) method. 
     In addition, the analysis method for diagnosis is selected from the group that includes but not limited to conventional polymerase chain reaction (C-PCR: conventional PCR), nested polymerase chain reaction (N-PCR: nested PCR), multiple polymerase chain reaction, real-time polymerase chain reaction, real-time quantitative polymerase chain reaction, and loop-mediated isothermal amplification (LAMP), but is not limited thereto. 
     The subject suspected of having anaplasmosis is one who have been infected or suspected of having being infected with  Anaplasma phagocytophilum , and may be selected from domestic animals such as ticks, rats, wild animals, cattle, pigs, sheep, goats, deer, horses, companion animals such as dogs and cats, and humans. 
     The biological sample may be a body fluid or secretion of the subject and examples thereof include, but are not limited to, blood, serum, plasma, lymph, cerebrospinal fluid, tissue fluid, or the like, or urine, tears, saliva, milk, vomit, feces, and the like, which are secretions from the body, and the biological sample may preferably be blood. 
     Hereinafter, the present disclosure will be described in more detail with reference to examples. The examples are provided only for illustration of the present disclosure, and should not be construed as limiting the scope of the present disclosure. 
     EXAMPLE 1 
     Production of Primers and Probes for Detecting and Diagnosing Anaplasmosis 
     While performing research to discover a novel biomarker enabling detection of anaplasmosis quickly, accurately, and with high sensitivity, the present inventors visited the Chosun University Hospital. They chose P 44  as a target gene that can be used as a biomarker for anaplasmosis diagnosis from among multiple-copy genes that exhibit differences in expression from normal subjects in blood obtained from patients diagnosed with anaplasmosis and infected with the  A. phagocytophilum  strain, and produced primers and probes capable of specifically amplifying P 44  as shown in Table 1, and in particular, designed the primers and probes of the present disclosure shown in Table 1 so as to diagnose anaplasmosis with high sensitivity through only simple PCR. 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Assay 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Name 
                 Primer, Probes 
                 Base Sequence 
                 Position 
                 Length 
                 Tm 
                 SEQ ID No. 
                 Product 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Ana P44 
                 Sense Primer 
                 GGATGGAAAGAGTGTAAAG 
                 90 
                 19 
                 59.1 
                 3 
                 154 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 CTCGTAACCAATCTCAAG 
                 243 
                 18 
                 58.5 
                 4 
                   
               
               
                 Ana P44 
                 Anti-sense Probe 
                 CACCACCAATACCATAACCAACACTG 
                 214 
                 26 
                 69 
                 16 
                   
               
               
                   
               
               
                 Ana P44 
                 Sense Primer 
                 GGATGGAAAGAGTGTAAAG 
                 90 
                 19 
                 59.1 
                 3 
                 154 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 CTCGTAACCAATCTCAAG 
                 243 
                 18 
                 58.5 
                 4 
                   
               
               
                 Ana P44 
                 Anti-sense Probe 
                 TACCATAACCAACACTGCCTTCCATA 
                 205 
                 26 
                 69 
                 17 
                   
               
               
                   
               
               
                 Ana P44 
                 Sense Primer 
                 GGATGGAAAGAGTGTAAAG 
                 90 
                 19 
                 59.1 
                 3 
                 154 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 CTCGTAACCAATCTCAAG 
                 243 
                 18 
                 58.5 
                 4 
                   
               
               
                 Ana P44 
                 Anti-sense Probe 
                 CAATACCATAACCAACACTGCCTTCC 
                 208 
                 26 
                 69.1 
                 18 
                   
               
               
                   
               
               
                 Ana P44 
                 Sense Primer 
                 GGATGGAAAGAGTGTAAAG 
                 90 
                 19 
                 59.1 
                 3 
                 154 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 CTCGTAACCAATCTCAAG 
                 243 
                 18 
                 58.5 
                 4 
                   
               
               
                 Ana P44 
                 Anti-sense Probe 
                 CCAATACCATAACCAACACTGCCTTC 
                 209 
                 26 
                 69.1 
                 19 
                   
               
               
                   
               
               
                 Ana P44 
                 Sense Primer 
                 GGATGGAAAGAGTGTAAAG 
                 90 
                 19 
                 59.1 
                 3 
                 154 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 CTCGTAACCAATCTCAAG 
                 243 
                 18 
                 58.5 
                 4 
                   
               
               
                 Ana P44 
                 Anti-sense Probe 
                 ATACCATAACCAACACTGCCTTCCATA 
                 206 
                 27 
                 69.1 
                 20 
                   
               
               
                   
               
               
                 Ana P44 
                 Sense Primer 
                 GGATGGAAAGAGTGTAAAG 
                 90 
                 19 
                 59.1 
                 3 
                 154 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 CTCGTAACCAATCTCAAG 
                 243 
                 18 
                 58.5 
                 4 
                   
               
               
                 Ana P44 
                 Anti-sense Probe 
                 TACCATAACCAACACTGCCTTCCA 
                 205 
                 24 
                 69.2 
                 21 
                   
               
               
                   
               
               
                 Ana P44 
                 Sense Primer 
                 GCTATGGAAGGCAGTGTTGG 
                 178 
                 20 
                 55.98 
                 5 
                 73 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 TGAAGCGCTCGTAACCAATC 
                 231 
                 20 
                 55.77 
                 6 
                   
               
               
                 Ana P44 
                 Anti-sense Probe 
                 AGCTCAACCCTGGCACCACCA 
                 207 
                 21 
                 63.17 
                 22 
                   
               
               
                   
               
               
                 Ana P44 
                 Sense Primer 
                 ACAGTCCAGCGTTTAGCAAG 
                 8 
                 20 
                 55.59 
                 7 
                 190 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 CCAACACTGCCTTCCATAGC 
                 178 
                 20 
                 55.98 
                 8 
                   
               
               
                 Ana P44 
                 sense Probe 
                 TGACTGGAACACTCCTGATCCTCGGA 
                 126 
                 26 
                 62.7 
                 23 
                   
               
               
                   
               
               
                 Ana P44 
                 Sense Primer 
                 CCTGATCCTCGGATTGGGTT 
                 139 
                 20 
                 56.16 
                 9 
                 81 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 CCTGGCACCACCAATACCAT 
                 200 
                 20 
                 57.02 
                 10 
                   
               
               
                 Ana P44 
                 Anti-sense Probe 
                 CCAACACTGCCTTCCATAGCTACAAGC 
                 171 
                 27 
                 62.02 
                 24 
                   
               
               
                   
               
               
                 Ana P44 
                 Sense Primer 
                 CCTGATCCTCGGATTGGGTT 
                 139 
                 20 
                 56.16 
                 9 
                 117 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 GGTCTTGAAGCGCTCGTAAC 
                 236 
                 20 
                 56.45 
                 11 
                   
               
               
                 Ana P44 
                 Anti-sense Probe 
                 AGCTCAACCCTGGCACCACCA 
                 207 
                 21 
                 63.17 
                 25 
                   
               
               
                   
               
               
                 Ana P44 
                 Sense Primer 
                 TATTGGTGGTGCCAGGGTT 
                 204 
                 19 
                 55.97 
                 12 
                 156 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 AGGTTATCAGTCTGCCCAGT 
                 340 
                 20 
                 55.02 
                 13 
                   
               
               
                 Ana P44 
                 Anti-sense Probe 
                 ACCCTTGGTCTTGAAGCGCTCGT 
                 239 
                 23 
                 63.22 
                 26 
                   
               
               
                   
               
               
                 Ana P44 
                 Sense Primer 
                 ACAAGTTTGACTGGAACACTCC 
                 119 
                 22 
                 55.47 
                 14 
                 101 
               
               
                 Ana P44 
                 Anti-sense Primer 
                 CCTGGCACCACCAATACCAT 
                 200 
                 20 
                 57.02 
                 15 
                   
               
               
                 Ana P44 
                 Anti-sense Probe 
                 CCAACACTGCCTTCCATAGCTACAAGC 
                 171 
                 27 
                 62.02 
                 27 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 2 
     Detection Sensitivity Analysis of the Present Disclosure Using Primers and Probe for Detecting Anaplasmosis 
     A test was performed to determine whether or not the primer and probe for detecting anaplasmosis of the present disclosure devised in Example 1 can quickly detect anaplasmosis with high sensitivity. 
     For this purpose, first, among patients who visited Chosun University Hospital from 2016 to 2017, blood from 15 patients diagnosed with anaplasmosis and blood from 15 patients diagnosed with a disease other than anaplasmosis were selected for testing. 
     Anaplasmosis was defined as a case in which the amount of an antibody against  A. phagocytophilum  increased more than fourfold in immunofluorescence antibody assay (IFA), and the positive control used herein was blood collected from 15 confirmed patients. In addition, the negative control used herein was blood obtained from 15 patients in whom an antibody was not detected by immunofluorescence antibody assay (IFA) and who were diagnosed with diseases other than anaplasmosis. Each of the blood samples obtained above was centrifuged, a buffy coat was collected therefrom, genomic genes were extracted therefrom, and PCR was performed using the primers or probes of the present disclosure shown in Table 1 to analyze the diagnostic potential and sensitivity of anaplasmosis. In addition, regarding comparative groups, PCR analysis was performed on 16S rRNA (GenBank: CP000235), ankA (GenBank: AF020521) and groEL-STG (GenBank: CP000235) genes. These genes are known to be conventional anaplasmosis diagnostic markers as template DNA. Using specific primers to compare the detection sensitivity of P 44 , a novel biomarker identified in the present disclosure, as well as the primers and probes for detecting the same. 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 PCR method 
                 Groel C-PCR 
                 AnkA PCR 
                 16S PCR 
                 MSP2 C-PCR 
                 P44 C-PCR 
                 16S N-PCR 
                 P44 Q-PCR 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Case 
                 Control 
                 Case  
                 Control 
                 Case 
                 Control 
                 Case  
                 Control 
                 Case 
                 Control 
                 Case 
                 Control 
                 Case 
                 Control 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Test positive 
                 6 
                 0 
                 6 
                 0 
                 6 
                 0 
                 6 
                 0 
                 11 
                 0 
                 11 
                 0 
                 15 
                 0 
               
               
                 Test negative 
                 9 
                 15 
                 9 
                 15 
                 8 
                 15 
                 9 
                 15 
                 4 
                 15 
                 4 
                 15 
                 0 
                 15 
               
               
                 Total 
                 15 
                 15 
                 15 
                 15 
                 14 
                 15 
                 15 
                 15 
                 15 
                 15 
                 15 
                 15 
                 15 
                 15 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Sensitivity, % (95% Cl) 
                 40(17-67) 
                 40(17-67) 
                 43( 9-70) 
                 43( 9-70) 
                 73(45-91) 
                 73(45-91) 
                 100(75-100) 
               
               
                 Specificity, % (95% Cl)  
                 100(75-100) 
                 100(74-100) 
                 100(74-100) 
                 100(75-100) 
                 100(75-100) 
                 100(75-100) 
                 100(75-100) 
               
               
                 PPV, % (95% Cl) 
                 100(52-100) 
                 100(52-100) 
                 100(52-100) 
                 100(52-100) 
                 100(68-100) 
                 100(68-100) 
                 100(75-100) 
               
               
                 NPV, % (95% Cl) 
                 63(41-80) 
                 63(41-80) 
                 65(43-83) 
                 65(43-83) 
                 79(54-93) 
                 79(54-93) 
                 100(75-100) 
               
               
                 Time 
                 3-4h 
                 3-4h 
                 3-4h 
                 3-4h 
                 3-4h 
                 6-7h 
                 1-2h 
               
               
                   
               
            
           
         
       
     
     In addition, the respective primer sequences used for the test are shown in Table 3 below, and PCR was respectively performed under the conditions described in Tables 4 and 5 below. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                   
                   
                   
                   
                   
                 Product 
               
               
                   
                   
                   
                   
                 SEQ ID 
                 size 
               
               
                 PCR 
                 Primer 
                 Primer Name 
                 Primer Sequence 
                 NO. 
                 (bp) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 groEL N-PCR 
                 1st 
                 GRO607F 
                 GAAGATGCWGTWGGWTGTACKGC 
                 28 
                 688 
               
               
                 1 st  PCR 
                 Forward 
                   
                   
                   
                   
               
               
                   
                 1st Reverse 
                 GRO1294R 
                 AGMGCTTCWCCTTCWACRTCYTC 
                 29 
                   
               
               
                   
               
               
                 groEL N-PCR 
                 2nd 
                 GRO677F 
                 ATTACTCAGAGTGCTTCTCARTG 
                 30 
                 445 
               
               
                 2 nd  PCR/C-PCR 
                 Forward 
                   
                   
                   
                   
               
               
                   
                 2nd 
                 GRO1121R 
                 TGCATACCRTCAGTYTTTTCAAC 
                 31 
                   
               
               
                   
                 Reverse 
                   
                   
                   
                   
               
               
                   
               
               
                 ankA N-PCR 
                 1st 
                 ANK-F1 
                 GAAGAAATTACAACTCCTGAAG 
                 32 
                 705 
               
               
                 1 st  PCR 
                 Forward 
                   
                   
                   
                   
               
               
                   
                 1st Reverse 
                 ANK-R1 
                 CAGCCAGATGCAGTAACGTG 
                 33 
                   
               
               
                   
               
               
                 ankA N-PCR 
                 2nd 
                 ANK-F2 
                 TTGACCGCTGAAGCACTAAC 
                 34 
                 664 
               
               
                 2 nd  PCR/C-PCR 
                 Forward 
                   
                   
                   
                   
               
               
                   
                 2nd 
                 ANK-R2 
                 ACCATTTGCTTCTTGAGGAG 
                 35 
                   
               
               
                   
                 Reverse 
                   
                   
                   
                   
               
               
                   
               
               
                 16s N-PCR 
                 1st 
                 AE1-F 
                 AAGCTTAACACATGCAAGTCGAA 
                 36 
                 1406 
               
               
                 1 st  PCR 
                 Forward 
                   
                   
                   
                   
               
               
                   
                 1st Reverse 
                 AE1-R 
                 AGTCACTGA CCCAACCTTAAATG 
                 37 
                   
               
               
                   
               
               
                 16s N-PCR 
                 2nd 
                 EE-3 
                 GTCGAACGGATTATTCTTTATAGCTTGC 
                 38 
                 926 
               
               
                 2 nd  PCR/C-PCR 
                 Forward 
                   
                   
                   
                   
               
               
                   
                 2nd 
                 EE-4 
                 CCCTTCCGTTAAGAAGGATCTAATCTCC 
                 39 
                   
               
               
                   
                 Reverse 
                   
                   
                   
                   
               
               
                   
               
               
                 msp2(P44) C- 
                 Forward 
                 msp2F 
                 ATGTCCATGGCTATAGTCATGGCTG 
                 40 
                 430 
               
               
                 PCR 
                 Reverse 
                 msp2R 
                 ACCTCGAGTTAAGCTAACTCCTTAGCT 
                 41 
                   
               
               
                   
               
               
                 msp2 N-PCR 
                 1st 
                 Anapmsp21F 
                 TTATGATTAGGCCTTTGGGCATG 
                 42 
                 1079 
               
               
                 1 st  PCR 
                 Forward 
                   
                   
                   
                   
               
               
                   
                 1st Reverse 
                 Anapmsp21R 
                 TCAGAAAGATACACGTGCGCCC 
                 43 
                   
               
               
                   
               
               
                 msp2 N-PCR 
                 2nd 
                 Anapmsp22F 
                 GGTTACATAAGGGCCGCAAAGGTG 
                 44 
                 467 
               
               
                 2 nd  PCR 
                 Forward 
                   
                   
                   
                   
               
               
                   
                 2nd 
                 Anapmsp22R 
                 CCGGCGCATGTGTAAGGTGAAA 
                 45 
                   
               
               
                   
                 Reverse 
                   
                   
                   
                   
               
               
                   
               
               
                 P44 Q-PCR 
                 Forward 
                 anaP44 90F 
                 GGATGGAAAGAGTGTAAAG 
                 46 
                 153 
               
               
                   
                 Reverse 
                 anaP44 243R 
                 CTCGTAACCAATCTCAAG 
                 47 
                   
               
               
                   
                 Probe 
                 anaP44 anti- 
                 [TET]CACCACCAATACCATAACCAACACT 
                 48 
                   
               
               
                   
                   
                 214P 
                 [BHQ1] 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                   
                 groEL N-PCR 
                 groEL N-PCR 
                 ankA N-PCR 
                 ankA N-PCR 
               
               
                   
                 lstPCR 
                 2ndPCR/C-PCR 
                 lstPCR 
                 2ndPCR/C-PCR 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Step 
                 Temperature 
                 Time 
                 Temperature 
                 Time 
                 Temperature  
                 Time 
                 Temperature 
                 Time 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 95 
                 5 m 
                 95 
                 5 m 
                 95 
                 5 m 
                 95 
                 5 m 
               
               
                 2 
                 95 
                 30 s 
                 95 
                 3 s 
                 95 
                 3 s 
                 95 
                 30 s 
               
               
                 3 
                 54 
                 30 s 
                 50 
                 3 s 
                 53 
                 30 s 
                 52 
                 30 s 
               
               
                 4 
                 72 
                 90 s 
                 72 
                 60 s 
                 72 
                 60 s 
                 72 
                 60 s 
               
               
                 5 
                 Step 2 
                 35 cycle 
                 Step 2 
                 5 cycle 
                 Step 2 
                 35 cycle 
                 Step2 
                 5 cycle 
               
               
                 6 
                 72 
                 5 m 
                 95 
                 30 s 
                 72 
                 5 m 
                 95 
                 5m 
               
               
                 7 
                   
                   
                 53 
                 30 s 
                   
                 54 
                 30 s 
                   
               
               
                 8 
                   
                   
                 72 
                 60 s 
                   
                 72 
                 30 s 
                   
               
               
                 9 
                   
                   
                 Step 6 
                 5 cycle 
                   
                 Step 6 
                 60 s 
                   
               
               
                 10 
                   
                   
                 95 
                 30 s 
                   
                 72 
                 5 m 
                   
               
               
                 11 
                   
                   
                 57 
                 30 s 
                   
                   
                   
                   
               
               
                 12 
                   
                   
                 72 
                 60 s 
                   
                   
                   
                   
               
               
                 13 
                   
                   
                 Step 10 
                 25 cycle 
                   
                   
                   
                   
               
               
                 14 
                   
                   
                 72 
                 5 m 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
             
            
               
                   
                   
                 16s N-PCR 
                   
               
               
                   
                 16s N-PCR 
                 2 nd  PCR 
                 msp2(P44) 
               
               
                   
                 1 st  PCR 
                 /C-PCR 
                 C-PCR 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Step 
                 Temperature 
                 Time 
                 Temperature 
                 Time 
                 Temperature 
                 Time 
               
               
                 1 
                 95 
                 5m 
                 95 
                 5m 
                 95 
                 5 m 
               
               
                 2 
                 95 
                 30 s 
                 95 
                 30 s 
                 95 
                 30 s 
               
               
                 3 
                 59 
                 3O s 
                 i56 
                 30 s 
                 56 
                 30 s 
               
               
                 4 
                 72 
                 90 s 
                 72 
                 60 s 
                 72 
                 60 s 
               
               
                 5 
                 Step 2 
                 35 cycle 
                 Step 2 
                 30 cycle 
                 Step 2 
                 35  
               
               
                   
                   
                   
                   
                   
                   
                 cycle 
               
               
                 6 
                 72 
                 5 m 
                 72 
                 5m 
                 72 
                 5 m 
               
            
           
           
               
               
               
               
            
               
                   
                 msp2 N-PCR 
                 msp2 N-PCR 
                 P44 
               
               
                   
                 1st PCR 
                 2nd PCR  
                 Q-PCR 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Step 
                 Temperature 
                 Time 
                 Temperature 
                 Time 
                 Temperature 
                 Time 
               
               
                 1 
                 95 
                 5 m 
                 95 
                 5m 
                 ps 
                 5 m 
               
               
                 2 
                 95 
                 30 s 
                 95 
                 30 s 
                 95 
                 5 s 
               
               
                 3 
                 54 
                 30 s 
                 57 
                 i3O s 
                 51 
                 5 s 
               
               
                 4 
                 72 
                 60 s 
                 72 
                 60 s 
                 Scan 
                 TET 
               
               
                 5 
                 Step 2 
                 35 cycle 
                 Step 2 
                 35 cycle 
                 Step 2 
                 45  
               
               
                   
                   
                   
                   
                   
                   
                 cycle 
               
               
                 6 
                 72 
                 5 m 
                 72 
                 5 m 
                 25 
                 1 m 
               
               
                   
               
            
           
         
       
     
     As shown in Table 2 and  FIG. 1 , the analysis revealed that PCR amplification products were not observed in any of the 15 negative control group samples, whereas PCR amplification products were detected in the blood of patients diagnosed with anaplasmosis. However, it took 3 to 7 hours to obtain detection results for target genes (16S rRNA, ankA, and groEL-STG) used as comparative groups, whereas it took 1 to 2 hours to detect P44, which was a novel biomarker, using the primer of the present disclosure. 
     In addition, the diagnostic sensitivity of anaplasmosis, obtained through detection of P 44 , was 100% when the primer devised in the present disclosure was used, indicating that sensitivity and accuracy are very high. In contrast, it was found that the diagnostic sensitivity of conventionally used target genes (16S rRNA, ankA, groEL-STG) was low. 
     In addition, it was found that when C-PCR was performed using the P 44  gene of the present disclosure, the analysis time was considerably shorter than when N-PCR was performed using other target genes. When real-time PCR (Q-PCR) was performed on P 44  gene, 100% sensitivity was obtained, which has much higher sensitivity and specificity than markers and detection primers that have been developed and used to date, indicating that the P 44  gene may accurately detect and diagnose anaplasmosis. 
     As previously stated, P 44 , which is a novel biomarker for diagnosing anaplasmosis according to the present disclosure, is a multicopy gene that exists in a large number of copies in the  Anaplasma phagocytophilum  genome, allowing detection of  Anaplasma phagocytophilum  infection with high sensitivity using only a small amount of DNA compared to conventional diagnostic marker genes. In addition, the primer set or probe for detecting and amplifying P 44  according to the present disclosure is capable of quickly and simply detecting anaplasmosis with high specificity and sensitivity, and is thus useful for early diagnosis of anaplasmosis. 
     Although the preferred embodiments of the present disclosure have been disclosed, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the disclosure as disclosed in the accompanying claims. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present disclosure is defined by the claims rather than the aforementioned description, and all differences falling within the scope of equivalents thereto should be construed as falling within the scope of the present disclosure.