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Multiplex detection of short nucleic acids

A technology of nucleotides and target nucleic acids, which is applied in the field of multiple detection of short nucleic acids, and can solve the problems of specificity reduction and other issues

Pending Publication Date: 2020-04-21
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, specific amplification of short nucleic acid sequences is challenging
Amplification primers are often in the range of 15-25 nucleotides, and shorter complementary sequences can result in reduced specificity

Method used

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  • Multiplex detection of short nucleic acids
  • Multiplex detection of short nucleic acids
  • Multiplex detection of short nucleic acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1: Use in Multiplex Assays to Improve Specificity and Signal Strength

[0133] Presently published results show that use of a single forward primer complementary to a portion of all reverse transcription products (e.g., spanning arm, step and / or loop sequences from the RT primer) results in little or no non-specific amplification . For a schematic diagram of the primers, see Figure 1A and Figure 1B . Figure 2A and Figure 2B Results from RT-qPCR run with target-specific forward primers (4-plex-spefwd) and universal forward primers (4-plex-unifwd) were compared. Figure 2A and Figure 2B The no RNA negative control in shows a significantly higher signal with the target-specific forward primer compared to the universal primer. Also, the signals of RNA-positive samples were not as strong in reactions with target-specific forward primers compared to those signals using universal primers. The four target RNA molecules are miR-39 (external control), miR-21 ...

Embodiment 2

[0134] Example 2: Probe modification reduces cross-reactivity in multiplex assays

[0135] Probes can cross-react when the target RNA molecule has a similar sequence at the 3' end. One option for addressing specificity in multiplex reactions is to combine RT primers for a panel of miRNAs in an RT reaction. Then, an aliquot of cDNA was amplified using a mixture of forward and reverse PCR primers for the same set of miRNA targets (RT products) along with miRNA target-specific probes in one PCR reaction. The probes in this case target the sequence between the forward and reverse primers of the second strand cDNA and are labeled with different fluorophores for each miRNA target for specific detection. In this design, the probes share the same 5' end, targeting arm, stem and / or loop sequence, followed by 8-10 target-specific nucleotides. For some miRNAs with very similar 3' sequences, this approach is problematic because the probes differ by only a few nucleotides near the 3' e...

Embodiment 3

[0140] Embodiment 3: comparison with prior art

[0141] We compared the results of the two-tailed RT primer approach disclosed herein for a 4-target multiplex reaction with those from a single target assay commercially available from ABI for the same miRNA target. The four miRNA targets are miR126, miR21, miR423 and miR39. The table below shows the Ct values ​​for titrated RT-PCR reactions using the multiplex, two-tailed RT primer method (upper table) and the singleplex ABI assay (lower table).

[0142] RNA mix cDNA cps / rxn Log 10 miR126 miR21 miR423 miR39 E7 2E+06 6.30 19.88 19.95 20.73 21.54 E6 2E+05 5.30 23.38 23.48 24.47 25.10 E5 2E+04 4.30 27.18 27.32 28.21 28.81 E4 2E+03 3.30 30.87 31.01 31.85 32.56 E3 2E+02 2.30 34.36 34.64 35.69 36.27 E2 2E+01 1.30 37.29 38.28 38.67 37.80 Min RTNTC 38.52 36.99 39.81 37.78 SLOPE -3.53 -3.69 -3.63 -3.69 RSQ 0.9985 0.99...

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Abstract

Provided herein are methods and compositions for performing multiplex RT-PCR to amplify short nucleic acids.

Description

[0001] Background of the invention [0002] Non-invasive sample collection (eg, blood, plasma, saliva, urine) is becoming more common, motivated by the ease and reduced cost of sample collection. Nucleic acid biomarkers in liquid biopsy samples are typically short, eg, less than 250 nucleotides in length. Examples of disease-associated biomarkers in liquid biopsy samples are miRNA molecules, several of which have been found at abnormal levels in various disease states. miRNA molecules are typically 16-30 nucleotides in length. [0003] Biomarker nucleic acids are generally quite rare in liquid biopsy samples, so amplification of the biomarker nucleic acids is beneficial to increase the sensitivity of detection. However, specific amplification of short nucleic acid sequences is challenging. Amplification primers are often in the range of 15-25 nucleotides, and shorter complementary sequences can result in reduced specificity. Thus, although amplification and detection techniq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6853C12Q1/6809
CPCC12Q1/6809C12Q1/6853C12Q2521/107C12Q2525/155C12Q2525/207C12Q2525/301C12Q2537/143C12Q1/686C12Q1/6806C12Q2600/16C12N2310/3231C12N15/10
Inventor C.利特斯特H.B.德兰W.杨
Owner F HOFFMANN LA ROCHE & CO AG