Method for isothermal dna amplification starting from an rna template

A method of quantifying a ribonucleic acid (RNA) template from a sample comprising:

• reverse-transcribing the RNA to form a cDNA using a first reaction mixture comprising RNA template, at least one primer capable of hybridizing to the RNA template, a reverse transcriptase and deoxynucleoside triphosphates (dNTPs);
• and
• amplifying the cDNA to form an amplified product using a second reaction mixture comprising at least one strand displacement DNA polymerase, at least one inosine-containing primer and a nuclease that is capable of nicking DNA 3′ to an inosine residue of the primer;
• analysing a rate of synthesis of the amplification product to quantify the RNA template present in the sample,
• wherein the reverse transcribing and amplifying are achieved under isothermal condition without denaturing the cDNA template.

A method of quantifying a ribonucleic acid (RNA) template from a sample comprising:

• reverse-transcribing the RNA to form a cDNA using a first reaction mixture comprising RNA template, at least one primer capable of hybridizing to the RNA template, a reverse transcriptase and deoxynucleoside triphosphates (dNTPs);
• and
• amplifying the cDNA to form an amplification product using a second reaction mixture comprising at least one strand displacement DNA polymerase, at least one inosine-containing primer and a nuclease that is capable of nicking DNA 3′ to an inosine residue of the primer;
• and
• detecting the amplification product using a nucleic acid detection system,
• wherein the reverse transcribing and amplifying are achieved under isothermal condition without denaturing the cDNA template.

A method of amplifying a ribonucleic acid (RNA) template, comprising:

• reverse-transcribing the RNA template to cDNA;
• and
• amplifying the cDNA to form an amplified product without denaturing the cDNA,
• wherein the reverse transcription and amplification occurs under an isothermal condition and in a single reaction mixture comprising the RNA template, at least one primer for capable of hybridizing to RNA, a reverse transcriptase, a strand displacement DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and at least one inosine-containing primer and a nuclease that is capable of nicking DNA 3′ to an inosine residue of the primer.