Methods and compositions related thereto for detecting and identifying distinct species of nucleic acids from causative agents
Methods are described herein for detecting and identifying distinct species of nucleic acids, in a single container, for example, from a certain genus of infectious agents or otherwise causative agents comprising, for example, providing a forward PCR primer common to a homologous gene region between the distinct species, and providing a reverse PCR primer common to a homologous gene region between the distinct species, to thereby define a PCR target region amongst the species, and providing a first oligonucleotide probe specific to a nucleic acid sequence within the target region that is characteristic of a first species, providing a second oligonucleotide probe specific to a nucleic acid sequence within the target region that is characteristic of a second species, wherein the first and second oligonucleotide probes are each detectably labeled with distinctly different detectable labels, conducting a PCR reaction in the container by means of the primers to amplify the target region amongst the species, and detecting the distinct labels, thereby identifying distinct species of nucleic acids corresponding to distinct species of infectious agents. Methods are preferred, for example, wherein the infectious agent is a member of the Herpesviridae family.
1. A single vessel assay system with a sensitivity of 100% and a specificity of 100% for the simultaneous detection and distinct identification of HSV-1 and HSV-2, comprising:
(a) a single vessel;
(b) a single set of forward and reverse primers present in said single vessel targeting against glycoprotein B gene of herpes virus, said forward primer consists of SEQ ID NO: 5 and said reverse primer consists of SEQ ID NO: 6;
(c) a first probe present in said single vessel, said first probe consists of SEQ ID NO: 7 and selectively hybridizes to a HSV-1 amplicon produced by said single set of forward and reverse primers in a real-time polymerase chain reaction when HSV-1 is present; and
(d) a second probe present in said single vessel, said second probe consists of SEQ ID NO: 8 and selectively hybridizes to a HSV-2 amplicon produced by said single set of forward and reverse primers in said real-time polymerase chain reaction when HSV-2 is present, said first probe and said second probe each has a different detectable label, said hybridization of said first probe to said HSV-1 amplicon and said second probe to said HSV-2 amplicon provides the simultaneous detection and distinct identification of HSV-1 and HSV-2 in a single tube reaction having a sensitivity of 100% and a specificity of 100%.
2. The single vessel assay system of claim 1 , further comprising: (e) a pyrosequencing reagent, said pyrosequencing reagent is used for determining the nucleotide sequence of said HSV-1 amplicon and said HSV-2 amplicon.