Methods and reagents for detecting ebola virus
Probes and primers are disclosed for detecting EBOV nucleic acid in a sample. Methods are also disclosed that utilize these probes and primers, wherein the methods can be used to detect an EBOV in a sample to identify a subject with an EBOV infection.
1. A method for detecting an EBOV NP nucleic acid in a biological sample from a subject, comprising:
amplifying a DNA template produced from a reverse transcription reaction performed on nucleic acid containing RNA isolated from the biological sample by:
contacting the DNA template under conditions suitable for amplification with a set of forward and reverse primers that can be used to amplify an EBOV nucleic acid molecule comprising the sequence set forth as SEQ ID NO: 1, and
performing a polymerase chain reaction amplification to form an amplified DNA product; and
contacting the amplified DNA product with a probe that can hybridize to the amplified DNA product;
wherein hybridization of the probe to the amplified DNA product indicates that EBOV NP nucleic acid is present in the sample.
2. The method of claim 1 , wherein the polymerase chain reaction amplification comprises a real-time polymerase chain reaction (RT-PCT).
3. The method of claim 1 , wherein the set of primers comprises a forward primer comprising, consisting essentially of, or consisting of the nucleic acid sequence set forth as SEQ ID NO: 2 (NP2-F).
4. The method of claim 1 , wherein the set of primers comprises a reverse primer comprising, consisting essentially of, or consisting of the nucleic acid sequence set forth as SEQ ID NO: 3 (NP2-R).
5. The method of claim 1 , wherein the set of primers comprises a forward primer and a reverse primer comprising, consisting essentially of, or consisting of the nucleic acid sequences set forth as SEQ ID NO: 2 (NP2-F) and SEQ ID NO: 3 (NP2-R), respectively.
6. The method of claim 1 , wherein the set of primers comprises a pair of reverse primers comprising, consisting essentially of, or consisting of the nucleic acid sequences set forth as SEQ ID NOs: 15 (NP2-R1) and 16 (NP2-R2).
7. The method of claim 1 , wherein the set of primers comprises:
a forward primer comprising, consisting essentially of, or consisting of the nucleic acid sequence set forth as SEQ ID NO: 2 (NP2-F), and a pair of reverse primers comprising, consisting essentially of, or consisting of the nucleic acid sequences set forth as SEQ ID NOs: 15 (NP2-R1) and 16 (NP2-R2).
8. The method of claim 1 , wherein the probe comprises or consists of the nucleotide sequence set forth as SEQ ID NO: 4 (NP2-P), or the complement thereof.
9. The method of claim 1 , wherein the set of primers comprises a forward primer comprising, consisting essentially of, or consisting of the nucleic acid sequence set forth as SEQ ID NO: 2 (NP2-F), and a pair of reverse primers comprising, consisting essentially of, or consisting of the nucleic acid sequences set forth as SEQ ID NOs: 15 (NP2-R1) and 16 (NP2-R2); and the probe comprises or consists of the nucleotide sequence set forth as SEQ ID NO: 4 (NP2-P), or the complement thereof.
10. The method of claim 1 , wherein the set of primers comprises a forward primer consisting of the nucleic acid sequence set forth as SEQ ID NO: 2 (NP2-F), and a pair of reverse primers consisting of the nucleic acid sequences set forth as SEQ ID NOs: 15 (NP2-R1) and 16 (NP2-R2); and the probe consists of the nucleotide sequence set forth as SEQ ID NO: 4 (NP2-P), or the complement thereof.
11. A method for detecting an EBOV VP40 nucleic acid in a biological sample from a subject, comprising:
amplifying a DNA template produced from a reverse transcription reaction performed on nucleic acid containing RNA isolated from the biological sample by:
contacting the DNA template under conditions suitable for amplification with a set of forward and reverse primers that can be used to amplify an EBOV nucleic acid molecule comprising the sequence set forth as SEQ ID NO: 5, and
performing a polymerase chain reaction amplification to form an amplified DNA product; and
contacting the amplified DNA product with a probe that can hybridize to the amplified DNA product;
wherein hybridization of the probe to the amplified DNA product indicates that EBOV NP nucleic acid is present in the sample.
12. The method of claim 1 , further comprising detecting the amplified DNA product hybridized to the probe, wherein detecting hybridization of the probe to the amplified DNA product indicates that EBOV NP nucleic acid is present in the sample.
13. The method of claim 1 , further comprising providing the DNA template.
14. The method of claim 1 , wherein the probe is labeled with a detectable moiety that provides a detectable signal.
15. The method of claim 14 , wherein the probe is labeled with a terminally-linked fluorophore and a terminally-linked non-fluorescent quencher for use in a RT-PCR assay.
16. The method of claim 1 , wherein:
detecting the amplified DNA product hybridized to the probe comprises determining a threshold cycle (Ct) of the test amplification; and
determining if the probe is hybridized to the amplified DNA product comprises determining that the Ct of the test amplification is less than the Ct of a negative control RT-PCR amplification performed using the forward and reverse primers and the probe on a sample lacking EBOV nucleic acid.
17. The method of claim 16 , wherein the threshold cycle of the test amplification is 38 cycles or fewer.
18. The method of claim 1 , wherein the DNA template is produced by:
isolating the nucleic acid containing RNA from the biological sample; and
performing the reverse transcription reaction on the isolated nucleic acid to produce the DNA template.
19. The method of claim 1 , wherein the biological sample is a blood, serum, plasma, or urine sample.
20. The method of claim 1 , wherein the subject has or is at risk of an EBOV infection.
21. The method of claim 1 , wherein the method detects EBOV nucleic acid from at least 95% of EBOV isolates.
22. The method of claim 1 , further comprising identifying the subject as a subject with an EBOV infection if EBOV nucleic acid is detected in the biological sample.
23. The method of claim 22 , further comprising treating EBOV infection in the subject.
24. The method of claim 23 , comprising administering a therapeutically effective amount of an anti-Ebola agent to the subject.