Semi-pan-protozoal by quantitative PCR

• US 8,778,843 B1
• Filed: 03/15/2013
• Issued: 07/15/2014
• Est. Priority Date: 08/03/2011
• Status: Active Grant

• Current Assignee: Fry Laboratories, LLC
• Original Assignee: Fry Laboratories, LLC
• Inventors: Fry, Stephen E., Ellis, Jeremy
• Primary Examiner(s): Riggs, Larry
• Assistant Examiner(s): DINES, KARLA A
• Application Number:
  US13/834,441
• Time in Patent Office: 487 Days
• Field of Search: 506/7
• US Class Current: 506/7
• CPC Class Codes: C12Q 1/6893 for protozoa
• CPC Class Codes: C12Q 2600/158 Expression markers
• 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

Disclosed are compositions, kits, and methods for detecting, extracting, visualizing, and identifying a pathogenic protozoan. Quantitative real time polymerase chain reaction in connection with specifically designed oligonucleotide probes are used to detect a variety of pathogenic protozoans in patient samples.

Claims

1. A method for determining whether a sample contains or has an increased likelihood of containing a pathogenic protozoan comprising:

a) providing a vessel containing a reaction mixture, wherein the reaction mixture comprises at least one forward primer comprising SEQ ID NO;

6, at least one reverse primer comprising SEQ ID NO;

7, a nucleic acid target from the sample, and a pool of oligonucleotide probes selected from the group consisting of;

i) oligonucleotide probes comprising the sequences of SEQ ID NO;

4, SEQ ID NO;

8, and SEQ ID NO;

9;

ii) oligonucleotide probes comprising the sequences of SEQ ID NO;

10, SEQ ID NO;

12 and SEQ ID NO;

15;

iii) oligonucleotide probes comprising the sequences of SEQ ID NO;

11, SEQ ID NO;

13 and SEQ ID NO;

14; and

iv) oligonucleotide probes comprising the sequences of SEQ ID NO;

16 and SEQ ID NO;

17;

wherein the reaction mixture is capable of amplifying, by a multiplex quantitative real time polymerase chain reaction (qPCR), a segment of the nucleic acid target to produce an amplicon; and

wherein production of the amplicon is primed by the at least one forward primer and the at least one reverse primer;

b) incubating the vessel under conditions allowing production of the amplicon if the sample contains the pathogenic protozoan; and

c) determining that the sample contains the pathogenic protozoan or that the sample has an increased likelihood of containing the pathogenic protozoan if the amplicon is detected, or determining that the sample does not contain the pathogenic protozoan or that the sample does not have an increased likelihood of containing the pathogenic protozoan if the amplicon is not detected.

2. The method of claim 1, wherein the oligonucleotide probes further comprise a fluorophore and/or a quencher.

3. The method of claim 1, further comprising:

(d) detecting fluorescence from the oligonucleotide probes in the reaction mixture; and

(e) identifying the pathogenic protozoan using an alignment of a sequence of the oligonucleotide probes with a genomic sequence from the pathogenic protozoan.

4. The method of claim 3, wherein the alignment indicates at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity between the sequence of the oligonucleotide probe and the genomic sequence.

5. The method of claim 1, wherein the pathogenic protozoan is selected from the group consisting of Protomyxzoa spp., Sarcocystis spp., Cyclophora spp., Eimeria spp., Goussia spp., Entomoeba histolytica, Acanthamoeba castellanii, Balamuthia mandrillaris, Trichomonas spp., Trypanosoma spp., Leishmania spp, Pneumocystis pneumonia, Naegleria fowleri, Giardia intestinalis, Blastocystis hominis, Babesia microti, Cryptosporidium spp., Cyclospora cayetanensis, Toxoplasma gondii, and Theileria spp.

6. The method of claim 5, wherein the Protomyxzoa spp. is Protomyxzoa rheumatica.

7. The method of claim 5, wherein the Cryptosporidium spp. is selected from Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium meleagridis, and Cryptosporidium muris.

8. The method of claim 5, wherein the Trichomonas spp. is selected from Trichomonas tenas, Trichomonas hominis, and Trichomonas vaginalis.

9. The method of claim 5, wherein the Trypanosoma spp. is selected from Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi and Trypanosoma brucei.

10. The method of claim 5, wherein the Leishmania spp. is selected from Leishmania donovani, Leishmania tropica, and Leishmania braziliensis.

11. The method of claim 5, wherein the Theileria spp. is selected from Theileria lawrenci and Theileria parva.

12. A diagnostic kit used to determine whether a sample contains or has an increased likelihood of containing a pathogenic protozoan comprising:

at least one forward primer comprising SEQ ID NO;

6 and at least one reverse primer comprising SEQ ID NO;

7;

a pool of oligonucleotide probes selected from the group consisting of;

i) oligonucleotide probes comprising the sequences of SEQ ID NO;

4, SEQ ID NO;

8, and SEQ ID NO;

9;

ii) oligonucleotide probes comprising the sequences of SEQ ID NO;

10, SEQ ID NO;

12, and SEQ ID NO;

15;

iii) oligonucleotide probes comprising the sequences of SEQ ID NO;

11, SEQ ID NO;

13, and SEQ ID NO;

14; and

iv) oligonucleotide probes comprising the sequences of SEQ ID NO;

16 and SEQ ID NO;

17;

wherein the oligonucleotide probes further comprise a fluorophore and/or a quencher;

an indication of a result of the presence of a nucleic acid from a pathogenic protozoan;

and instructions for using the kit;

wherein the kit utilizes multiplex quantitative real time PCR (qPCR).