Provided herein are reagents and methods for comprehensively enriching potential variants within targeted regions, named Amplicon Comprehensive Enrichment (ACE). The sequence variants enriched can include single nucleotide polymorphisms (SNPs), single nucleotide variants, or small insertions and deletions. Embodiments include procedures for integration with real-time polymerase chain reaction, next generation sequencing (NGS), and long-read sequencing.

Provided herein are reagents and methods for comprehensively enriching potential variants within targeted regions, named Amplicon Comprehensive Enrichment (ACE). The sequence variants enriched can include single nucleotide polymorphisms (SNPs), single nucleotide variants, or small insertions and deletions. Embodiments include procedures for integration with real-time polymerase chain reaction, next generation sequencing (NGS), and long-read sequencing.

A one-step nested PCR primers set and a kit modified with locked nucleic acid for detecting African swine fever virus are provided, relating to the field of molecular biology. It includes an outer primer pair, an inner primer pair and a probe. Upstream and downstream primer sequences of the outer primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively. Upstream and downstream primer sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively. A sequence of the probe is shown in SEQ ID NO: 5. Based on the principle of nested PCR, the Tm value and stability of outer primers are improved by designing two pairs of nested primers and simultaneously modifying the outer primers with locked nucleic acid, and two independent circular nested amplification are performed, which has high sensitivity and specificity, easiness in operation and less cross contamination.

A one-step nested PCR primers set and a kit modified with locked nucleic acid for detecting African swine fever virus are provided, relating to the field of molecular biology. It includes an outer primer pair, an inner primer pair and a probe. Upstream and downstream primer sequences of the outer primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively. Upstream and downstream primer sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively. A sequence of the probe is shown in SEQ ID NO: 5. Based on the principle of nested PCR, the Tm value and stability of outer primers are improved by designing two pairs of nested primers and simultaneously modifying the outer primers with locked nucleic acid, and two independent circular nested amplification are performed, which has high sensitivity and specificity, easiness in operation and less cross contamination.

Methods according to aspects of the disclosure, compositions and kits therefore, include at least one, two, or three sets of amplification primers and hydrolysis probes with at least two separate corresponding readouts per set. According to aspects of the present disclosure, the at least two hydrolysis probes and associated pair of primers of each set are directed to opposite strands of an amplification product of the set. According to aspects of the present disclosure, one of the two hydrolysis probes in each set is directed to a first strand of the amplification product and therefore has a sequence complementary to the first strand of the amplification product and the second of the two hydrolysis probes in the set is directed to the second strand of the amplification product and therefore has a sequence complementary to the second strand of the amplification product.

Methods according to aspects of the disclosure, compositions and kits therefore, include at least one, two, or three sets of amplification primers and hydrolysis probes with at least two separate corresponding readouts per set. According to aspects of the present disclosure, the at least two hydrolysis probes and associated pair of primers of each set are directed to opposite strands of an amplification product of the set. According to aspects of the present disclosure, one of the two hydrolysis probes in each set is directed to a first strand of the amplification product and therefore has a sequence complementary to the first strand of the amplification product and the second of the two hydrolysis probes in the set is directed to the second strand of the amplification product and therefore has a sequence complementary to the second strand of the amplification product.

The present invention relates to the detection of a target nucleic acid sequence by a PTOCE (PTO Cleavage and Extension) assay. The present invention detects a target nucleic acid sequence in which the PTO (Probing and Tagging Oligonucleotide) hybridized with the target nucleic acid sequence is cleaved to release a fragment and the fragment is hybridized with the CTO (Capturing and Templating Oligonucleotide) to form an extended duplex, followed by detecting the presence of the extended duplex. The extended duplex provides signals (generation, increase, extinguishment or decrease of signals) from labels indicating the presence of the extended duplex and has adjustable T.sub.m value, which are well adoptable for detection of the presence of the target nucleic acid sequence.

The present invention relates to the detection of a target nucleic acid sequence by a PTOCE (PTO Cleavage and Extension) assay. The present invention detects a target nucleic acid sequence in which the PTO (Probing and Tagging Oligonucleotide) hybridized with the target nucleic acid sequence is cleaved to release a fragment and the fragment is hybridized with the CTO (Capturing and Templating Oligonucleotide) to form an extended duplex, followed by detecting the presence of the extended duplex. The extended duplex provides signals (generation, increase, extinguishment or decrease of signals) from labels indicating the presence of the extended duplex and has adjustable T.sub.m value, which are well adoptable for detection of the presence of the target nucleic acid sequence.

The present disclosure relates to a method of detecting methylation of target DNA in a multiplex manner and a composition for detecting methylation of target DNA, and more particularly to a method for detecting methylation of target DNA, comprising: constructing an oligonucleotide, which comprises a target-specific sequence capable of binding complementarily to the target DNA and a universal primer that does not bind complementarily to the target DNA; linearly amplifying the target DNA for linear target enrichment by using the oligonucleotide as a primer; and amplifying the linearly amplified target DNA using an oligonucleotide, which is capable of binding complementarily to the linearly amplified target DNA, a universal primer, and a probe.

The present disclosure relates to a method of detecting methylation of target DNA in a multiplex manner and a composition for detecting methylation of target DNA, and more particularly to a method for detecting methylation of target DNA, comprising: constructing an oligonucleotide, which comprises a target-specific sequence capable of binding complementarily to the target DNA and a universal primer that does not bind complementarily to the target DNA; linearly amplifying the target DNA for linear target enrichment by using the oligonucleotide as a primer; and amplifying the linearly amplified target DNA using an oligonucleotide, which is capable of binding complementarily to the linearly amplified target DNA, a universal primer, and a probe.