Primers, probes and methods for sensitive, specific detection and monitoring of HIV-1 and HCV
Primers and probes for detecting an RNA virus, including HIV, HIV-1 subtypes of the M and O groups, and HCV, in a sample. The primers and probes can be used for monitoring the efficacy of anti-retroviral treatment in a subject infected with HIV and/or HCV, and for detecting acute HIV-1 infection, and/or acute HCV infection, in a subject. Included are inner, middle and outer primers that can be used in PCR, including triple nested PCR in a single tube. The methods are highly sensitive and specific, allowing for detection of as few as 4 copies of virus in a sample.
1. A method for detecting HCV in a sample comprising: isolating RNA in a sample, submitting the isolated RNA to reverse transcription PCR, contacting the resulting reverse transcription PCR product with a set of primers, and submitting the resulting mixture to one or more nucleic acid amplification reactions, wherein the set of primers comprises the collection of oligonucleotides comprising: one or more forward primers selected from the group consisting of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, complementary sequences thereof, active fragments thereof, and combinations thereof; one or more reverse primers selected from the group consisting of SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, complementary sequences thereof, active fragments thereof, and combinations thereof; and one or more probes selected from the group consisting of SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:26, complementary sequences thereof, active fragments thereof, and combinations thereof.
2. A method for detecting HCV in a sample comprising: isolating RNA in a sample, submitting the isolated RNA to reverse transcription PCR, contacting the resulting reverse transcription PCR product with a set of outer primers, and submitting the resulting mixture to a first nucleic acid amplification reaction, wherein the primers comprise a combination of oligonucleotides comprising an outer, middle and inner set of primers wherein the outer primers comprise SEQ ID NO:16, SEQ ID NO:17 or active fragments thereof; the middle primers comprise SEQ ID NO:18, SEQ ID NO: 19 or active fragments thereof; the inner primers comprise SEQ ID NO:20, SEQ ID NO:21 or active fragments thereof.
3. The method of claim 2 , further comprising contacting a product from the first nucleic acid amplification reaction with a set of middle primers and submitting the resulting mixture to a second nucleic acid amplification reaction.
4. The method of claim 3 , further comprising contacting a product from the second nucleic acid amplification reaction with a set of inner primers and submitting the resulting mixture to a third nucleic acid amplification reaction.
5. The method of claim 4 , further comprising contacting a product from the third nucleic amplification reaction with a probe for quantitation by real time PCR; wherein the probe is selected from the group consisting of SEQ ID NO:22, or active fragments thereof.
6. A method for detecting HCV in a sample comprising: isolating RNA in a sample, submitting the isolated RNA to reverse transcription PCR, contacting the resulting reverse transcription PCR product with a set of outer primers, and submitting the resulting mixture to a first nucleic acid amplification reaction, wherein the primers comprises a combination of oligonucleotides comprising an outer, middle and inner set of primers wherein the outer primers comprise SEQ ID NO:16, SEQ ID NO:23 or active fragments thereof; the middle primers comprise SEQ ID NO:18, SEQ ID NO:24 or active fragments thereof; the inner primers comprise SEQ ID NO:20, SEQ ID NO:21 or active fragments thereof.
7. The method of claim 6 , further comprising contacting a product from the first nucleic acid amplification reaction with a set of middle primers and submitting the resulting mixture to a second nucleic acid amplification reaction.
8. The method of claim 7 , further comprising contacting a product from the second nucleic acid amplification reaction with a set of inner primers and submitting the resulting mixture to a third nucleic acid amplification reaction.
9. The method of claim 8 , further comprising contacting a product from the third nucleic amplification reaction with the probe for quantitation by real time PCR; wherein the probe is selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:26, and active fragments thereof.
10. The method of claim 4 , wherein the first nucleic acid amplification reaction is performed at a temperature of about 65-71° C., the second nucleic acid amplification reaction is performed at a temperature of about 60-64° C., and the third nucleic acid amplification reaction is performed at a temperature of about 50-55° C.
11. The method of claim 4 , wherein the method is carried out in a single tube.
12. The method of claim 1 , wherein the sample contains less than 5 copies of the RNA virus.
13. A method for monitoring the efficacy of anti-retroviral treatment in a subject infected with HCV, the method comprising:
(a) subjecting a sample obtained from the subject to the method of claim 8 ; and
(b) determining whether the sample contains fewer copies of the virus per milliliter than a predetermined threshold;
wherein a reduction in copies of the virus per milliliter sample is indicative of effective anti-retroviral treatment.
14. The method of claim 13 , wherein the predetermined threshold is an amount detected in a prior sample obtained from the subject at a previous time point.
15. The method of claim 13 , wherein the predetermined threshold is 5 copies of virus per milliliter sample.
16. A method for detecting acute early HCV infection in a subject, the method comprising:
(a) subjecting a sample obtained from the subject to the method of claim 1 ; and
(b) determining whether the sample contains a detectable amount of HCV RNA;
wherein the presence of a detectable amount of HCV RNA is indicative of acute early HCV infection.
17. The method of claim 16 , wherein the sample is blood, plasma, serum, saliva, urine, cerebral spinal fluid, milk, cervical secretions, semen, tissue, or cell cultures.
18. The method of claim 1 , wherein the one or more forward primers comprise SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or active fragments thereof; and the one or more reverse primers comprise SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or active fragments thereof; and the probe comprises SEQ ID NO:22, or an active fragment thereof.
19. The method of claim 18 , wherein the set of primers comprises an outer, middle and inner set of primers, wherein the outer primers comprise SEQ ID NO:16, SEQ ID NO:17 or active fragments thereof; the middle primers comprise SEQ ID NO:18, SEQ ID NO: 19 or active fragments thereof; the inner primers comprise SEQ ID NO:20, SEQ ID NO:21 or active fragments thereof; and the one or more probes selected from the group consisting of SEQ ID NO:22, or active fragments thereof.
20. The method of claim 1 , wherein the one or more forward primers comprise SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, or active fragments thereof; and the one or more reverse primers comprise SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:21, or active fragments thereof; and the one or more probes comprise SEQ ID NO:25, SEQ ID NO:26, or active fragments thereof.
21. The method of claim 1 , wherein the collection of oligonucleotides comprises an outer, middle and inner set of primers wherein the outer primers comprise SEQ ID NO:16, SEQ ID NO:23 or active fragments thereof; the middle primers comprise SEQ ID NO:18, SEQ ID NO:24 or active fragments thereof; the inner primers comprise SEQ ID NO:20, SEQ ID NO:21 or active fragments thereof; and one or more probes comprise SEQ ID NO:25, SEQ ID NO:26, or active fragments thereof.
22. The method of claim 8 , wherein the first nucleic acid amplification reaction is performed at a temperature of about 65-71° C., the second nucleic acid amplification reaction is performed at a temperature of about 60-64° C., and the third nucleic acid amplification reaction is performed at a temperature of about 50-55° C.
23. The method of claim 8 , wherein the method is carried out in a single tube.
24. The method of claim 4 , wherein the sample contains less than 5 copies of the RNA virus.
25. The method of claim 8 , wherein the sample contains less than 5 copies of the RNA virus.