Methods and compositions for detecting Zika virus
Provided herein is a highly sensitive and robust method for Zika detection in semen, as well as related compositions. The method can include: (a) extracting nucleic acids from a human semen sample; (b) detecting Zika virus nonstructural protein 5 (NS5) mRNA using real-time reverse-transcription polymerase chain reaction (rRT-PCR); and (c) simultaneously, in the rRT-PCR, detecting human beta-actin mRNA as positive control.
1. A method for detecting Zika virus, comprising:
(a) extracting nucleic acids from a human semen sample;
(b) detecting a 73-nucleotide region in Zika virus nonstructural protein 5 (NS5) mRNA using real-time reverse-transcription polymerase chain reaction (rRT-PCR), wherein said 73-nucleotide region consists of g.9389 to g.9461 of GenBank Accession number KU501215; and
(c) simultaneously, in the rRT-PCR, detecting human beta-actin mRNA as positive control, comprising using a forward primer, a reverse primer and a TAQMAN® probe having the nucleic acid sequences of SEQ ID NOs: 7, 8 and 9, respectively, for detecting the human beta-actin mRNA.
2. The method of claim 1 , wherein the nucleic acids are RNA.
3. The method of claim 1 , wherein the human semen sample is fresh, refrigerated, frozen or cryopreserved.
4. The method of claim 1 , wherein step (b) further comprises providing:
a forward primer that is 15-25 nucleotides long and targets a region located within g.9389 to g.9414 of GenBank Accession number KU501215;
a reverse primer that is 15-25 nucleotides long and targets a region located within g.9436 to g.9461 of GenBank Accession number KU501215; and
a TAQMAN® probe that is 18-30 nucleotides long and targets a region located within g.9404 to g.9446 of GenBank Accession number KU501215.
5. The method of claim 4 , wherein the forward primer, the reverse primer and the probe consist of the nucleic acid sequences of SEQ ID NOs: 1, 2 and 3, respectively.
6. A method for detecting two or more viruses, comprising:
(a) extracting nucleic acids from a human semen sample;
(b) subjecting a first portion of the nucleic acids to real-time reverse-transcription polymerase chain reaction (rRT-PCR) for detecting Zika virus nonstructural protein 5 (NS5) mRNA, while simultaneously, in the rRT-PCR, detecting human beta-actin mRNA as a first positive control; and
(c) subjecting a second portion of the nucleic acids to real-time PCR for detecting one or more DNA viruses, while simultaneously, in the real-time PCR, detecting human Amelogenin gene as a second positive control.
7. The method of claim 6 , wherein the nucleic acids are a mixture of RNA and DNA.
8. The method of claim 6 , wherein step (b) comprises detecting a 73-nucleotide region from g.9389 to g.9461 or a 91-nucleotide region from g.9142 to g.9232 of GenBank Accession number KU501215.
9. The method of claim 8 , further comprising providing:
a forward primer that is 15-25 nucleotides long and targets a region located within g.9389 to g.9414 or g.9142 to g.9167 of GenBank Accession number KU501215;
a reverse primer that is 15-25 nucleotides long and targets a region located within g.9436 to g.9461 or g.9207 to g.9232 of GenBank Accession number KU501215; and
a TAQMAN® probe that is 18-30 nucleotides long and targets a region located within g.9404 to g.9446 or g.9157 to g.9217 of GenBank Accession number KU501215.
10. The method of claim 9 , wherein the forward primer has the nucleic acid sequence of SEQ ID NO: 1 or 4, the reverse primer has the nucleic acid sequence of SEQ ID NO: 2 or 5, and the probe has the nucleic acid sequence of SEQ ID NO: 3 or 6.
11. The method of claim 6 , wherein the one or more DNA viruses are selected from cytomegalovirus (CMV), herpes simplex virus (HSV) 1 & 2, and human papillomavirus (HPV) strains 16 & 18.
12. The method of claim 11 , further comprising providing:
a forward primer, a reverse primer and a TAQMAN® probe having the nucleic acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, for detecting CMV;
a forward primer, a reverse primer and a TAQMAN® probe having the nucleic acid sequences of SEQ ID NOs: 13, 14 and 15, respectively, for detecting HSV 1;
a forward primer, a reverse primer and a TAQMAN® probe having the nucleic acid sequences of SEQ ID NOs: 16, 17 and 18, respectively, for detecting HSV 2;
a forward primer, a reverse primer and a TAQMAN® probe having the nucleic acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, for detecting HPV 16; and/or
a forward primer, a reverse primer and a TAQMAN® probe having the nucleic acid sequences of SEQ ID NOs: 22, 23 and 24, respectively, for detecting HPV 18.
13. The method of claim 6 , wherein step (c) comprises using a forward primer, a reverse primer and a TAQMAN® probe having the nucleic acid sequences of SEQ ID NOs: 25, 26 and 27, respectively, for detecting the human Amelogenin gene.