Microsporidia Detection System and Method

• US 20130035248A1
• Filed: 05/21/2012
• Published: 02/07/2013
• Est. Priority Date: 05/20/2011
• Status: Abandoned Application

• Current Assignee: Phthisis Diagnostics, Inc. (Microbiologics, Inc.)
• Original Assignee: Phthisis Diagnostics, Inc. (Microbiologics, Inc.)
• Inventors: Icenhour, Crystal R.
• Application Number:
  US13/476,355
• Publication Number:
  US 20130035248A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

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.

Claims

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.