Method of detection of classical swine fever

A method for detecting the presence of classical swine fever virus (CSFV) ribonucleic acids in a biological sample, said method comprising:

• (i) reverse transcribing ribonucleic acids obtained from the biological sample to obtain cDNA;
• (ii) contacting said cDNA with a first forward primer, a first reverse primer, and a DNA polymerase to produce a first PCR product, wherein the first forward primer consists of SEQ ID NO:
• 2, and the first reverse primer consists of SEQ ID NO:
• 4;
• (iii) hybridizing to the first PCR product a first nucleic acid probe consisting of SEQ ID NO:
• 3;
• (v) contacting an internal control ribonucleic acid derived from SEQ ID NO:
• 10 with at least two second primers capable of hybridizing to the internal control ribonucleic acid;
• and
• (iv) detecting hybridization of said first nucleic acid probe to said first PCR product, wherein hybridization of said first nucleic acid probe to said first PCR product is indicative of the presence of CSFV nucleic acids in the biological sample.

A method for detecting the presence of classical swine fever virus (CSFV) ribonucleic acids in a biological sample, said method comprising:

• (i) obtaining a biological sample, said biological sample comprising ribonucleic acids;
• (ii) reverse transcribing said ribonucleic acids to obtain cDNA;
• (iii) contacting said cDNA with a first forward primer, a first reverse primer, and a DNA polymerase to produce a first PCR product, wherein the first forward primer consists of SEQ ID NO:
• 2, and the first reverse primer consists of SEQ ID NO:
• 4;
• (iv) hybridizing to the first PCR product a first nucleic acid probe consisting of SEQ ID NO:
• 3;
• (v) contacting an internal control ribonucleic acid derived from SEQ ID NO:
• 10 with a second forward primer and a second reverse primer to produce a second PCR product, wherein the second forward primer hybridizes to a target site corresponding to nucleotides 637 to 656 of SEQ ID NO:
• 10 or a complementary strand thereof, and the second reverse primer hybridizes to a target site corresponding to nucleotides 750 to 768 of SEQ ID NO:
• 10 or a complementary strand thereof;
• (vi) hybridizing to the second PCR product a second nucleic acid probe that hybridizes to a target site corresponding to nucleotides 703 to 724 of SEQ ID NO:
• 10 or a complementary strand thereof;
• and
• (vii) detecting hybridization of said first nucleic acid probe to said first PCR product, wherein hybridization of said first nucleic acid probe to said first PCR product is indicative of the presence of CSFV nucleic acids in the biological sample.

A method of quantifying classical swine fever virus (CSFV) viral load in a first isolated sample, comprising:

• (i) contacting said first isolated sample with a first forward primer, a first reverse primer, and a DNA polymerase to produce a first PCR product, wherein the first forward primer consists of SEQ ID NO:
• 2, and the first reverse primer consists of SEQ ID NO:
• 4, and hybridizing a first nucleic acid probe consisting of SEQ ID NO:
• 3 to the first PCR product, wherein hybridizing the first nucleic acid probe consisting of SEQ ID NO:
• 3 to the first PCR product provides a first detectable signal, and measuring the intensity of said first detectable signal;
• (ii) contacting said first sample with a second nucleic acid probe, wherein the second probe hybridizes to an internal control ribonucleic acid derived from SEQ ID NO:
• 10, wherein hybridization of the second nucleic acid probe to the internal control ribonucleic acid derived from SEQ ID NO:
• 10 provides a second detectable signal, and measuring the intensity of said second detectable signal;
• (iii) contacting a control sample having a known CSFV viral load with a nucleic acid probe consisting of SEQ ID NO:
• 3 to provide a third detectable signal and measuring the intensity of said third signal;
• and
• (iv) comparing the intensity of the first signal to the intensity of the second signal and the third signal, wherein said comparing indicates the quantity of classical swine fever virus viral load in the first isolated sample.