A method for discriminating between live and dead microbes in a sample, by discriminating between transcriptionally-active and inert microbial nucleic acid sequences in the sample. In particular, the method is based on the comparison of levels of nucleotide substitution in a sample cultured in presence of an RNA-labelling agent. Also, a diagnosis method of microbial infections in a subject; and methods of assessing the risk of contamination of a sample, implementing the method for discriminating between live and dead microbes in a sample.

A method for generating region-specific amplification templates of a biological sample includes adding first oligonucleotide constructs and second oligonucleotide constructs to the biological sample. Each first or second oligonucleotide construct comprises a first or a second photoremovable cage molecule. The method further includes synthesising a complementary first strand from a template bound to target binding regions of each first oligonucleotide construct or each second oligonucleotide construct, scanning a first region of interest of the biological sample with a first focused light beam and a second region of interest of the biological sample with a second focused light beam to form uncaged first oligonucleotide constructs in the first region of interest and uncaged second oligonucleotide constructs in the second region of interest, synthesising a complementary second strand to form first amplification templates originating from the first region of interest and second amplification templates originating from the second region of interest.

A method for generating region-specific amplification templates of a biological sample includes adding first oligonucleotide constructs and second oligonucleotide constructs to the biological sample. Each first or second oligonucleotide construct comprises a first or a second photoremovable cage molecule. The method further includes synthesising a complementary first strand from a template bound to target binding regions of each first oligonucleotide construct or each second oligonucleotide construct, scanning a first region of interest of the biological sample with a first focused light beam and a second region of interest of the biological sample with a second focused light beam to form uncaged first oligonucleotide constructs in the first region of interest and uncaged second oligonucleotide constructs in the second region of interest, synthesising a complementary second strand to form first amplification templates originating from the first region of interest and second amplification templates originating from the second region of interest.

This invention generally relates to a novel RNA/mRNA production and amplification method using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes as well as the associated mRNAs thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least an RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp), particularly coronaviral and hepatitis C viral (HCV) RdRp enzymes.

This invention generally relates to a novel RNA/mRNA production and amplification method using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes as well as the associated mRNAs thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least an RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp), particularly coronaviral and hepatitis C viral (HCV) RdRp enzymes.

A method for specifically and efficiently quantifying the expression of targeted RNA variants with specific terminal sequences suitable to identify multiple isoforms bearing complex heterogeneity in terminal sequences by hybridizing a 5′-Dbs-adapter to the 5′-end of target RNAs, wherein the 5′-Dbs-adapter has a stem-loop structure whose protruding 5′-end base-pairs with the 5′-end of target RNAs, and wherein the loop region of 5′-Dbs-adapter contains a base-lacking spacer which will terminate reverse transcription in a subsequent step; hybridizing a 3′Db-adapter to the 3′-end of target RNAs, wherein the 3′-Db-adapter has a stem-loop structure whose protruding 3′-end base-pairs with the 3′-end of target RNAs; ligating both adapters with target RNAs by RN12 ligation to form a “dumbbell-like” structure; and, amplifying and quantifying the ligation product by RT-PCR.

A method for specifically and efficiently quantifying the expression of targeted RNA variants with specific terminal sequences suitable to identify multiple isoforms bearing complex heterogeneity in terminal sequences by hybridizing a 5′-Dbs-adapter to the 5′-end of target RNAs, wherein the 5′-Dbs-adapter has a stem-loop structure whose protruding 5′-end base-pairs with the 5′-end of target RNAs, and wherein the loop region of 5′-Dbs-adapter contains a base-lacking spacer which will terminate reverse transcription in a subsequent step; hybridizing a 3′Db-adapter to the 3′-end of target RNAs, wherein the 3′-Db-adapter has a stem-loop structure whose protruding 3′-end base-pairs with the 3′-end of target RNAs; ligating both adapters with target RNAs by RN12 ligation to form a “dumbbell-like” structure; and, amplifying and quantifying the ligation product by RT-PCR.

The present invention is directed to compositions, methods and kits useful for the generation of nucleic acids from RNA templated and further nucleic acid amplification and detection. Specifically, the invention is directed to the generation and amplification of nucleic acids by reverse transcriptase polymerase chain reaction (RT-PCR). Provided are compositions and methods for improved amplification of nucleic acid molecules in a two-step, addition only RT-PCR procedure. The invention thus facilitates the rapid and efficient amplification of nucleic acid molecules and the detection and quantitation of RNA molecules, and is useful for a variety of research, industrial, medical and forensic purposes.

The present invention is directed to compositions, methods and kits useful for the generation of nucleic acids from RNA templated and further nucleic acid amplification and detection. Specifically, the invention is directed to the generation and amplification of nucleic acids by reverse transcriptase polymerase chain reaction (RT-PCR). Provided are compositions and methods for improved amplification of nucleic acid molecules in a two-step, addition only RT-PCR procedure. The invention thus facilitates the rapid and efficient amplification of nucleic acid molecules and the detection and quantitation of RNA molecules, and is useful for a variety of research, industrial, medical and forensic purposes.

This invention provides methods for near-instantaneously separating cells that have undergone RNA guided genome modifications into pre-templated instant partitions (PIPs) and using the PIPs to associate the guide RNAs with the gene expression level changes that resulted from the genome modification.