Microsporidia Detection System and Method
In various aspects, a multiplex primer set, a kit for performing an assay to detect microsporidia, and a method of identifying a microsporidia in a sample is provided. The multiplex PCR primer set including SEQ ID NOS 1-4. The kit includes a multiplex PCR primer set having SEQ ID NOS 1-4 and a set of probes having SEQ ID NOS 5-8. In the method a sample is obtained and a multiplex PCR assay is performed on the sample. A multiplex PCR primer set including SEQ ID NOS 1-4 is included in the multiplex PCR assay. The sample is determined to have the microsporidia in response to the multiplex PCR assay amplifying a target sequence associated with microsporidia.
1 . A multiplex PCR primer set comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
2 . The multiplex PCR primer set according to claim 1 , wherein the SEQ ID NO:1 and SEQ ID NO:2 are specific for Encephalitozoon intestinalis.
3 . The multiplex PCR primer set according to claim 1 , wherein the SEQ ID NO:3 and SEQ ID NO:4 are specific for Enterocytozoon bieneusi.
4 . A kit for performing an assay to detect microsporidia, the kit comprising:
a multiplex PCR primer set comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; and
a set of probes comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
5 . The kit according to claim 4 , wherein the SEQ ID NO:1 and SEQ ID NO:2 are specific for Encephalitozoon intestinalis.
6 . The kit according to claim 4 , wherein the SEQ ID NO:3 and SEQ ID NO:4 are specific for Enterocytozoon bieneusi.
7 . The kit according to claim 4 , further comprising a pair of primers specific for an internal control having the sequence of SEQ ID NO:9.
8 . The kit according to claim 7 , wherein the SEQ ID NO:3 and SEQ ID NO:4 are specific for the internal control having the sequence of SEQ ID NO:9.
9 . The kit according to claim 7 , further comprising a set of internal probes comprising SEQ ID NO:10 and SEQ ID NO:11.
10 . The kit according to claim 4 , further comprising a first dye being attached to the SEQ ID NO:6, wherein the first dye fluoresces at a wavelength of 605-620 nanometers (nm).
11 . The kit according to claim 4 , further comprising a second dye being attached to the SEQ ID NO:8, wherein the second dye fluoresces at a wavelength of 700-710 nm.
12 . The kit according to claim 4 , further comprising a third dye being attached to the SEQ ID NO:11, wherein the third dye fluoresces at a wavelength of 660-670 nm.
13 . A method further comprising of a multiplex real-time PCR assay amplifying a target associated with diagnosis of microsporidia.
14 . A method of identifying a microsporidia in a sample, the method comprising the steps of:
obtaining the sample;
performing a multiplex PCR assay on the sample and a multiplex PCR primer set comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; and
determining the sample contains the microsporidia in response to the multiplex PCR assay amplifying a target sequence associated with microsporidia.
15 . The method according to claim 14 , wherein the SEQ ID NO:1 and SEQ ID NO:2 are specific for Encephalitozoon intestinalis.
16 . The method according to claim 14 , wherein the SEQ ID NO:3 and SEQ ID NO:4 are specific for Enterocytozoon bieneusi.
17 . The method according to claim 14 , further comprising an internal control having the sequence of SEQ ID NO:9 included in the multiplex PCR assay.
18 . The method according to claim 17 , wherein the SEQ ID NO:3 and SEQ ID NO:4 are specific for the internal control having the sequence of SEQ ID NO:9.
19 . The method according to claim 17 , further comprising a set of internal probes comprising SEQ ID NO:10 and SEQ ID NO:11 included in the multiplex PCR assay.
20 . The method according to claim 14 , further comprising a set of probes comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 being included in the multiplex PCR assay.
21 . The method according to claim 20 , further comprising determining the patient is infected with Encephalitozoon intestinalis in response to detecting a first dye being attached to the SEQ ID NO:6, wherein the first dye fluoresces at a wavelength of 613 nanometers (nm).
22 . The method according to claim 20 , further comprising determining the patient is infected with Enterocytozoon bieneusi in response to detecting a second dye being attached to the SEQ ID NO:8, wherein the second dye fluoresces at a wavelength of 704 nm.
23 . The method according to claim 19 , further comprising determining the patient is infected with Enterocytozoon bieneusi in response to detecting a third dye being attached to the SEQ ID NO:11, wherein the third dye fluoresces at a wavelength of 665 nm.
24 . The method according to claim 14 , wherein the sample is obtained from a patient.
25 . The method according to claim 14 , wherein the sample is obtained from soil.
26 . The method according to claim 14 , wherein the sample is obtained from water.