Primer pair, kit and method for detecting anaplasma platys

Abstract

Primer pair, kit and method for detecting Anaplasma platys are disclosed. The primer pair includes a forward primer and a reverse primer, and the kit includes the primer pair and a probe. The forward primer has a sequence of SEQ ID NO: 1, the reverse primer has a sequence of SEQ ID NO: 2, and the probe has a sequence of SEQ ID NO: 3.

Claims

1. A method for detecting Anaplasma platys, the method comprising amplifying nucleic acid from Anaplasma platys using real-time polymerase chain reaction with a forward primer consisting of a sequence of 5-GTCGTAGCTTGCTATGATA-3 (SEQ ID NO: 1) and a reverse primer consisting of a sequence of 5-CCATACTACTAGGTAGATTCC-3 (SEQ ID NO: 2).

2. The method according to claim 1 wherein a probe consisting of a sequence of 5-CTCACCCGTCTGCCACTAAC-3 (SEQ ID NO: 3) is used for the real-time polymerase chain reaction.

3. The method according to claim 2 wherein the probe is labeled with a 5-reporter dye and a 3-quencher.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the corresponding positions of the forward primer, the reverse primer and the probe on the sequence of the 16S rRNA gene;

(2) FIG. 2 shows the DNA sequences of the forward primer, the reverse primer and the probe; and

(3) FIGS. 3A and 3B show the analysis of the amplification of the real-time PCR assay.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(4) The present invention will now be described more specifically with reference to the following embodiments. It is to be noted that the following descriptions of preferred embodiments of this invention are presented herein for purpose of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.

(5) The present invention utilizes real-time polymerase chain reaction (Real-time PCR), also called quantitative polymerase chain reaction (Q-PCR), with probe-based detection for detecting Anaplasma platys. In Real-time PCR, the specific forward and reverse primers and probe hybridize to the DNA target of Anaplasma platys, wherein the probe is labeled with a 5-reporter dye and a 3-quencher. During PCR amplification, the probe is cleaved and the reporter dye and quencher are separated, so that the resulting increase in fluorescence can be detected. In an embodiment, the reporter dye is FAM fluorescence, and the quencher is BHQ1 group.

(6) The DNA target for this assay is a variable region of the 16S rRNA gene (GenBank accession number: KP939260.1) that contains sequence that is species-specific for Anaplasma platys. PCR primers and probe are designed using Primer3 and chosen on the basis of GC content and lack of hairpin structures. FIG. 1 shows the corresponding positions of the forward primer, the reverse primer and the probe on the sequence of the variable region of the 16S rRNA gene (SEQ ID NO: 4). As shown in FIG. 1, the forward primer starts at position 3, the probe starts at position 44, and the reverse primer starts at position 74. This primers and probe combination is predicted to amplify the DNA of Anaplasma platys strains with an amplicon size of 72-bp. FIG. 2 shows the DNA sequences of the forward primer, the reverse primer and the probe, wherein the forward primer (SEQ ID NO: 1) includes 19-mer, the reverse primer (SEQ ID NO: 2) includes 21-mer, and the probe (SEQ ID NO: 3) includes 20-mer.

(7) To ascertain the specificity of the PCR primers and the probe for Anaplasma platys, the primer pair, including the forward primer and the reverse primer, and the probe are checked by Primer-BLAST from NCBI, and the blast result shows that no other similar species have 100% same fragment compare to the primer pair and the probe of the present invention. The result demonstrates that the specificity of the primer pair and the probe is quite high, and the primer pair and the probe can be only used to amplify and detect the 16S rRNA gene of Anaplasma platys.

(8) Therefore, the present invention provides a primer pair for detecting Anaplasma platys, comprising a forward primer having a sequence of 5 -GTCGTAGCTTGCTATGATA-3 (SEQ ID NO: 1) and a reverse primer having a sequence of 5-CCATACTACTAGGTAGATTCC-3 (SEQ ID NO: 2). The present invention also provides a kit for detecting Anaplasma platys, comprising a forward primer having a sequence of 5-GTCGTAGCTTGCTATGATA-3 (SEQ ID NO: 1), a reverse primer having a sequence of 5-CCATACTACTAGGTAGATTCC-3 (SEQ ID NO: 2) and a probe having a sequence of 5-CTCACCCGTCTGCCACTAAC-3 (SEQ ID NO: 3). On the other hand, the present invention also provides a method for detecting Anaplasma platys, the method comprising amplifying nucleic acid from Anaplasma platys using real-time polymerase chain reaction with a forward primer having a sequence of 5-GTCGTAGCTTGCTATGATA-3 (SEQ ID NO: 1), a reverse primer having a sequence of 5-CCATACTACTAGGTAGATTCC-3 (SEQ ID NO: 2) and a probe having a sequence of 5-CTCACCCGTCTGCCACTAAC-3 (SEQ ID NO: 3).

(9) In some other embodiments, since the primer pair of the present invention is specific to Anaplasma platys, all the sequence located between the forward primer and the reverse primer may be used as the probe sequence, and thus, the probe sequence is not limited to the aforesaid sequence. Further, the probe can be designed to hybridize to any strand of the DNA, so both the complementary sequences at the same location can be used as the probe sequence. Therefore, the complementary sequence of the aforesaid probe sequence may also be used as the probe sequence for detecting Anaplasma platys.

(10) The following describes an example of the method for detecting Anaplasma platys of the present invention.

(11) First, DNA is extracted from 200 l of EDTA-preserved whole blood using the QIAamp DNA blood Mini kit for blood protocol (Qiagen) and eluted in 100 l of elution buffer. Then the real-time PCR assay is performed on the Bio-Rad real-time PCR machine (CFX96). The PCR reaction mixture includes 10 l of KAPA Fast probe universal master mix, 250 nM of forward and reverse primers and 250 nM of probe, wherein the forward primer has a sequence of 5-GTCGTAGCTTGCTATGATA-3 (SEQ ID NO: 1), the reverse primer has a sequence of 5-CCATACTACTAGGTAGATTCC-3 (SEQ ID NO: 2) and the probe has a sequence of 5-CTCACCCGTCTGCCACTAAC-3 (SEQ ID NO: 3). 3 l extracted DNA template is added to each reaction in a total volume of 20 l. Cycling conditions are as follows: 95 C. for 3 min, followed by 40 cycles of denaturation at 95 C. for 3 sec, and annealing/extension at 60 C. for 20 sec.

(12) An Anaplasma platys-positive control is constructed by cloning the partial 16S rRNA gene fragment into a vector (RBC Cloning System). A series of seven 10-fold dilutions are prepared from this recombinant plasmid DNA (10, 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6 and 10.sup.7 copies/l). The dilution series are analyzed in duplicate to determine the lower limit of Anaplasma platys DNA detection and the linearity and efficiency of amplification of this real-time PCR assay.

(13) FIGS. 3A and 3B show the analysis of the amplification of the real-time PCR assay. FIG. 3A shows the amplification curve of different copies of plasmid samples, which reveals that the assay has high sensitivity. FIG. 3B shows the assay has good linearity with an R.sup.2 of 0.98, which is very close to the theoretical optimum of 1.0. Therefore, the assay could be expanded as a quantitative assay to estimate gene copy number in clinical samples.

(14) In conclusion, the present invention provides a method for detecting Anaplasma platys using real-time PCR with specific primer pair and probe. The method of the present invention has advantage of high sensitivity, and should allow the detection of low Anaplasma platys in subclinically infected cases. Moreover, diagnosis in early stage or acute phase is very critical for Anaplasma platys treatment; some studies show that when dogs are treated in the acute phase of anaplasmosis, they improve quickly, within 24-48 hours, and their prognosis is good when the whole courses of therapy are administered. The method of the present invention further has advantage of high specificity, which is able to specifically differentiate Anaplasma platys with other tick-borne pathogens and is very helpful for vets to choose the optimal treatment program. In addition, in recurrent disease after treatment or failure after treatment, the method of the present invention could determine if the original diagnosis was incorrect, so as to reduce risk of transfusion transmission by testing blood donors.

(15) While the invention has been described in terms of what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention needs not be limited to the disclosed embodiment. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.