Detection of microsporidial species using a quantitative real-time PCR assay

• US 20030215811A1
• Filed: 05/17/2002
• Published: 11/20/2003
• Est. Priority Date: 05/17/2002
• Status: Abandoned Application

• Current Assignee: Frank Schaefer, Jeffery Dean Hester, Lindquist H.D. Alan, Manju Varma
• Original Assignee: Frank Schaefer, Jeffery Dean Hester, Lindquist H.D. Alan, Manju Varma
• Inventors: Schaefer, Frank, Varma, Manju, Lindquist, H.D. Alan, Hester, Jeffery Dean
• Application Number:
  US10/147,322
• Publication Number:
  US 20030215811A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6893 for protozoa

Abstract

Microsporidial species can be detected in samples using real-time PCR dual-fluorescent assays with species-specific primer set and a dual fluorescent labeled hybridization probe. Cyclospora cayetanensis can be detected in samples using a real-time PCR dual-fluorescent assay with a primer set and dual fluorescent labeled hybridization probe.

Claims

1. A method for detecting microsporidial species in samples comprising:

a. degrading a dual-fluorescently labeled DNA hybridization probe and a set of species-specific primers during PCR amplification of regions in a gene located within the spores of interest; and

b. reading the fluorescent labels during the PCR process.

2. The method according to claim 1 wherein the microsporidial species is selected from the group consisting of Encephalitozoon hellen, Encephalitozoon, cuniculi, and Encephalitozoon intestinalis.

3. The method according to claim 2 wherein the gene is the 16S rRNA gene sequence of the Encephalitozoon species.

4. The method according to claim 1 wherein the dual-fluorescently labeled probe is labeled with a reporter fluorescent dye and a quencher fluorescent dye.

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

6. The method according to claim 3 wherein the dual-fluorescently labeled probe is labeled with a reporter fluorescent dye and a quencher fluorescent dye.

7. A method for detecting Cyclospora cayetanensis in samples comprising:

a. degrading a dual-fluorescently labeled species specific DNA hybridization probe and a primer set during PCR amplification of regions in a gene located within oocysts of interest; and

b. reading the fluorescent labeled during the PCR process.

8. The method according to claim 7 wherein the gene is the 18S rRNA sequence of Cyclospora cayetanensis.

9. The method according to claim 7 wherein the dual-fluorescently labeled probe is labeled with a reporter fluorescent dye and a quencher fluorescent dye.

10. The method according to claim 8 wherein the dual-fluorescently labeled probe is labeled with a reporter fluorescent dye and a quencher fluorescent dye.