The present invention provides a method for amplifying a sequence adjacent to a specific sequence, comprising the steps of: annealing a first forward primer to the specific sequence to synthesize a complementary strand; sequentially polymerically adding a first deoxynucleotide and a second deoxynucleotide to a 3′-end of the complementary strand; annealing a first reverse primer to a binding site between the 3′-end of the complementary strand and a polydeoxynucleotide strand composed of the first deoxynucleotide to synthesize a double-stranded DNA; performing a PCR with the double-stranded DNA as a template by using a second forward primer complementary to the specific sequence and a first reverse primer; and further performing a PCR by using a third forward primer complementary to the specific sequence and a second reverse primer.

A circular mRNA molecule possessing features resembling native mammalian mRNA demonstrates improved translation, while retaining the properties of an extremely long half-life inside cells. This circular mRNA is functional inside mammalian cells, being able to compete against native cellular mRNAs for the eukaryotic translation initiation machinery. The invention possesses additional RNA elements compared to a previous invention containing only an IRES element for successful in vitro or in vivo translation.

A circular mRNA molecule possessing features resembling native mammalian mRNA demonstrates improved translation, while retaining the properties of an extremely long half-life inside cells. This circular mRNA is functional inside mammalian cells, being able to compete against native cellular mRNAs for the eukaryotic translation initiation machinery. The invention possesses additional RNA elements compared to a previous invention containing only an IRES element for successful in vitro or in vivo translation.

The present invention provides methods for rapidly identifying an RNA viral infection using an isothermal nucleic acid amplification reaction that can be carried out extracted RNA in the context of a crude biological sample.

The present invention provides methods for rapidly identifying an RNA viral infection using an isothermal nucleic acid amplification reaction that can be carried out extracted RNA in the context of a crude biological sample.

Provided herein is a viral RNA detection system, utilizing the RNA-targeting properties of the optimized Cas13d enzyme, CasRx, to detect SARS-CoV-2 RNA, e.g., synthetic SARS-CoV-2 RNA. The system detects novel target sequences conserved within the actively evolving genome, to provide a panel of diagnostic target sites least likely to result in false negatives due to genomic variation. Successful detection of viral RNA through both a fluorescence-based readout assay as well as a rapid paper dipstick lateral flow assay requiring no specialized laboratory equipment was shown. Low viral titers can be detected within minutes following only minutes of sample processing.

Provided herein is a viral RNA detection system, utilizing the RNA-targeting properties of the optimized Cas13d enzyme, CasRx, to detect SARS-CoV-2 RNA, e.g., synthetic SARS-CoV-2 RNA. The system detects novel target sequences conserved within the actively evolving genome, to provide a panel of diagnostic target sites least likely to result in false negatives due to genomic variation. Successful detection of viral RNA through both a fluorescence-based readout assay as well as a rapid paper dipstick lateral flow assay requiring no specialized laboratory equipment was shown. Low viral titers can be detected within minutes following only minutes of sample processing.

A method of amplifying RNA template is provided. The method comprises reverse-transcribing a ribonucleic acid (RNA) template 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. The method is accomplished under an isothermal condition without denaturing the cDNA template. A method of quantifying RNA template in a sample and a method of detecting RNA template in a sample are also provided.

A method of amplifying RNA template is provided. The method comprises reverse-transcribing a ribonucleic acid (RNA) template 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. The method is accomplished under an isothermal condition without denaturing the cDNA template. A method of quantifying RNA template in a sample and a method of detecting RNA template in a sample are also provided.

The present invention relates to novel primers and sloppy molecular beacon and molecular beacon probes for amplifying segments from different genes in Mycobacterium tuberculosis for identifying the presence of M.tb DNA and/or resistance to anti-tuberculosis drugs.