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Non-competitive internal controls for use in nucleic acid tests

A non-competitive, internal control technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as inability to use multiple analysis

Inactive Publication Date: 2011-02-16
SIEMENS HEALTHCARE DIAGNOSTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of using a competitive internal control is that based on its structure, in particular, depending on the molar ratio, length, sequence and secondary structure of the nucleic acid fragments, the simultaneous amplification of two different nucleic acid fragments flanked by the same primer sites Increased risk of inhibition or enhancement of one or both products
Another disadvantage of competitive internal controls is that they cannot be used in multiplex assays, which screen for multiple targets in a single assay

Method used

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  • Non-competitive internal controls for use in nucleic acid tests
  • Non-competitive internal controls for use in nucleic acid tests
  • Non-competitive internal controls for use in nucleic acid tests

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Preparation of the MET IC DNA insert sequence

[0089] Genomic DNA was extracted from MET samples. DNA inserts were prepared by PCR on genomic DNA using the fragment primers in Table 1. The following sequence is the sequence (SEQ ID NO: 37) of the MET IC PCR product (213 bp). XhoI and SpeI restriction enzyme sites are underlined in bold.

[0090] 1 AGTAGTC CATGTGCA GGGATCCTGA CACGGTACTG

[0091] TCATCAG GTACACGT CCCTAGGACT GTGCCATGAC

[0092] 41 GAGGCAGGCA GGGCCGCCAT AAGAGCCATA GAGGAGGTTG

[0093] CTCCGTCCGT CCCGGCGGTA TTCTCGGTAT CTCCTCCAAC

[0094] 81 AGGGTGTTGT GACGCCCTTT GATATCTGCT CCGCAGCATC

[0095] TCCCACAACA CTGCGGGAAA CTATAGACGA GGCGTCGTAG

[0096] 121 AAAGCCAGAG ACAAATTACC CCTGGATAGG CCCCACCACG

[0097] TTTCGGTCTC TGTTTAATGG GGACCTATCC GGGGTGGTGC

[0098] 161 AACCACCCCT ACTGCCCGAG CCTGAAGGAG GTGCTCGGTG

[0099] TTGGTGGGGA TGACGGGCTC GGACTTCCTC CACGAGCCAC

[0100] 201AA CGA CGA

[0101] TT GCT GCT

Embodiment 2

[0103] Purified MET IC DNA Insert Sequence

[0104]The following sequence is the purified 195 bp dsDNA sequence (SEQ ID NO: 38) following restriction enzyme digestion at the sites identified above:

[0105] 1 TCGAGCATGT GCAGGGATCC TGACACGGTA CTGGAGGCAG

[0106] 41 GCAGGGCCGC CATAAGAGCC ATAGAGGAGG TTGAGGGTGT

[0107] 81 TGTGACGCCC TTTGATATCT GCTCCGCAGC ATCAAAGCCA

[0108] 121 GAGACAAATT ACCCCTGGAT AGGCCCCACC ACGAACCACC

[0109] 161 CCTACTGCCC GAGCCTGAAG GAGGTGCTCG GTGAA

Embodiment 3

[0111] MET IC DNA transcript sequence

[0112] A plasmid was prepared by ligating the purified MET IC DNA insert sequence of Example 2 into a purified vector fragment and adding the T7 promoter sequence. Purified vector fragments were isolated from Cloning vector (Invitrogen, Carlsbad, California). Plasmids are formed by matching the XhoI and SpeI cohesive ends of the DNA insert and the vector. attached figure 1 A schematic diagram of the cloning process is shown.

[0113] The resulting plasmid was linearized with XhoI and SpeI to generate the following 247bp MET ICDNA transcript sequence. The vector sequence is underlined in bold (SEQ ID NO: 39):

[0114] 1 CTC GAGCATGTGC

[0115] 41 AGGGATCCTG ACACGGTACT GGAGGCAGGC AGGGCCGCCA

[0116] 81 TAAGAGCCATAGAGGAGGTTGAGGGTGTTGTGACGCCCTT

[0117] 121 TGATATCTGC TCCGCAGCAT CAAAGCCAGA GACAAATTAC

[0118] 161 CCCTGGATAG GCCCCACCAC GAACCACCCC TACTGCCCGA

[0119] 201 GCCTGAAGGA GGTGCTCGGT GAACTAGT

[0120] 241

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Abstract

Provided are non-competitive internal controls for use in nucleic acid tests (NATs), which are obtained from the organisms Methanobacterium thermoautrophicum (MET) and Zea mays (Corn). The non-competitive internal controls have utility in DNA and RNA NATs selected from Influenza A, Influenza B, parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), respiratory syncytial virus type A (RSV A), RSV B, human metapneumovirus (hMPV), Chlamydia trachomatis (CT), and Neisseria gonorrhea (GC), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human Immunodeficiency Virus I (HIV-1), and Severe Acute Respiratory Syndrome (SARS).

Description

technical field [0001] The present application relates generally to tools for performing diagnostic assays, and more particularly to internal control sequences for nucleic acid testing (NAT) that do not compete with target nucleic acid sequences. Background of the invention [0002] In order to ensure that nucleic acid testing (NAT) is performed correctly, the analysis requires the presence of internal controls. In diagnosing NAT, the presence of internal controls ensures the integrity of the test. Specifically, by including an internal control in the NAT, samples that test positive for both the internal control and the target nucleic acid are true positives. Conversely, samples tested only for the internal control are true negatives, samples tested only for the target nucleic acid are true negatives, and samples with no detectable internal control or target are false negatives. [0003] The most commonly used diagnostic assay requiring the presence of an internal control ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6876C12Q2600/166C12Q1/6806C12Q2545/101
Inventor J·德特默X·蒋M·乐D·谢尔曼
Owner SIEMENS HEALTHCARE DIAGNOSTICS INC
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