Two-step Multiplex Nested Pcr
Two-step Multiplex Nested Pcr - Related Videos
03:20
Nested-PCR to Detect a Specific Viral Genomic Sequence
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2025
This video describes nested polymerase chain reaction, a technique that consists of two sequential PCR amplification processes using two primer sets. The first set of primers is intended to anneal to sequences upstream of the second set, resulting in selective amplification of specific gene sequences. This PCR is more sensitive and specific than a normal PCR and is widely used as a detection technique for various diseases.
10:15
Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
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Cited by 21 •
2011
An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.
03:41
Agarose Gel Electrophoresis of DNA Amplicons Post PCR: A Method to Analyze Products of Multiplex PCR and Evaluate PCR Reaction Success
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2025
In this video, we demonstrate the separation of bacterial PCR-amplified DNA using agarose gel electrophoresis. Agarose gel functions as a molecular sieve, enabling the negatively-charged DNA to migrate based upon their size under an applied electric field.
07:27
High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
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Cited by 13 •
2011
Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.
09:03
Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus
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Cited by 18 •
2013
We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.
FAQs
The second amplification uses primers positioned inside the products generated during the first PCR. A target must therefore be recognized by primer pairs in both rounds, which helps reduce detection of nonspecific products. This added selectivity is especially valuable when infectious-agent sequences or immune-related markers occur at low abundance within complex clinical or biological samples.
Multiplexing allows several primer pairs to amplify distinct genetic regions in the same initial reaction. Each pair contributes information about a different target, so one sample can support broader analysis than a single-target assay. This design is useful when researchers need to examine multiple infectious agents, immune-related markers, or genetically related organisms without consuming separate portions of the sample.
A single reaction can produce information about several genetic targets, while the nested second step adds focused re-amplification of selected products. Together, these features combine breadth with enhanced detection specificity. The resulting assay can help distinguish whether a sample contains one agent, multiple agents, or related genetic signatures, supporting more complete interpretation of complex biological material.
Specificity depends on two levels of primer recognition. The first primer pairs identify distinct regions for initial amplification, and the second-round primers bind within those initial products. Because the later reaction targets internal sequences rather than the surrounding sample background, the design can reduce nonspecific detection and improve confidence that an observed signal corresponds to the intended genetic target.
The workflow begins by performing a multiplex first PCR with several primer pairs, allowing distinct target regions to be amplified together. Products from that reaction then enter a second PCR in which primers located within selected initial products re-amplify the targets of interest. This sequential design preserves multiplex screening while adding a more selective detection stage.
It is useful when samples may contain low-abundance nucleic acids from infectious agents or immune-related markers. In infection studies, the assay can support pathogen identification and co-infection analysis. In immunology, its multiplex format can examine several relevant genetic targets from limited biological material, expanding the information obtained without requiring a separate assay for every target.
The combination of amplified targets can indicate which infectious agents or immune-related genetic markers are detectable in a sample. Comparing multiple signals may support co-infection analysis and help characterize genetically related organisms. Because the assay works with complex clinical or biological samples, it can provide a broader molecular profile than focusing on only one genetic target.