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PCR method of multiple primer, its reaction liquor and application for preparing detection reagent

A multiplex, reactive technique used in molecular biology to increase specificity and sensitivity and eliminate complexity

Inactive Publication Date: 2008-05-21
徐定邦 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The technical problem to be solved by the present invention is to provide a polymerase chain reaction method with multiple primers and its reaction solution and its application in the preparation of detection reagents, so as to break through the limitations of multiple primer design in the existing multiple PCR method and overcome the limitations of multiple primers in a single tube. The defect that PCR reactions cannot amplify multiple fragments of the same or different DNA sequences independently and simultaneously without affecting each other

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  • PCR method of multiple primer, its reaction liquor and application for preparing detection reagent
  • PCR method of multiple primer, its reaction liquor and application for preparing detection reagent
  • PCR method of multiple primer, its reaction liquor and application for preparing detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Comparison of ultra-low denaturing temperature multiplex PCR with conventional multiplex PCR.

[0029] Three pairs of primers were designed on the human actomyosin gene (GenBank No. BC016045), and their sequences and properties are listed in Tables 2 and 3, respectively.

[0030] Table 2. The primer sequence of Example 1

[0031]

[0032] Table 3. Primer properties of Example 1

[0033]

[0034]

[0035] Since the three pairs of genes target the same target gene and are relatively close to each other, unpaired amplification products will be formed under conventional multiplex PCR conditions, and their lengths and melting temperatures are shown in Table 4.

[0036] Table 4. Embodiment 1 Unpaired primer amplification product characteristics

[0037] unpaired primers

Amplified product length (bp)

Amplification product melting temperature (°C)

A01 forward primer and A02 reverse primer

360

87.9

A01 forward primer and A03 ...

Embodiment 2

[0044] Multiplex PCR with 6 pairs of primers.

[0045] The primer design is shown in Table 6.

[0046] The primer properties are listed in Table 7.

[0047] The characteristics of the amplification products with unpaired primers are shown in Table 8.

[0048] Table 6. Example 2 primer sequence

[0049]

[0050] Table 7. Example 2 Primer Characteristics

[0051]

[0052]

[0053] The base length of human actomyosin is less than 2000 bases, and 6 pairs of primers have the possibility of 15 kinds of unpaired combination amplification. The measured length and melting temperature of the amplified products are shown in Table 8.

[0054] Table 8. Embodiment 2 Unpaired primer amplification product characteristics

[0055]

[0056]

[0057] The melting temperatures of the six paired amplification products were all lower than 76.8°C, while the melting temperatures of the 15 unpaired amplification products were all higher than 80.9°C. Example 2 The reaction reagents a...

Embodiment 3

[0059] Design scheme and implementation conditions of ultra-low denaturation temperature multiplex PCR with 10 pairs of primers.

[0060] The Cyclin-D gene (GenBank No. NC_053056) was analyzed with the primer design software Oligo, and 10 pairs of primers with high stringency and the melting temperature of the amplified products were all lower than 81°C were searched. The positions and characteristics of the 10 pairs of primers and the characteristics of the 45 unpaired interference amplification products are listed in Table 9 and Table 10.

[0061] Table 10. 10 pairs of primers obtained by Cyclin D gene test

[0062] Primer pair number

[0063] Table 11. Test results of mutual interference products between each pair of primers

[0064] Primer pair number

[0065] 1

[0066] 5

[0067] The results show that the Tm value of the interference product is between 82.1-92.1°C, and most of them are between 83-87°C, which is obviously not i...

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Abstract

The present invention relates to a PCR method of multiple primer, its reaction liquid and its application in preparation of microbial detection reagent, specially relates to that in PCR process two denaturation temp. of 92-97 deg.C and 65-87 deg.C are adopted to respectively make initial 2 or 3 circulation or other subsequent circulation so as to overcome the defect of existent multiple PCR which can form non-specific product. Said invention utilizes the proper product design and adoption to two denaturation temperatures so as to obviously reduce the possibility of that non-target product early-synthesized by PCR can participate in subsequence. Said invention is applicable to preparation of single-tubule multiple PCR quick detection reagent, and has extensive application for detecting various viruses.

Description

technical field [0001] The invention relates to molecular biology technology, in particular to a polymerase chain reaction method, its reaction solution and its application in the preparation of detection reagents. technical background [0002] The classic polymerase chain reaction method was invented in the mid-1980s. It uses a forward primer and a reverse primer to amplify a specific gene segment. Soon, a multiplex PCR (Multiplex-PCR) method was developed in which two or more pairs of primers were used to simultaneously amplify several products in one reaction tube. Several primer pairs of current multiplex PCR are usually complementary to several different target gene sequences, which are carried on independent cDNAs, or carried on the same genomic DNA but separated from each other by a considerable distance. The current multiplex PCR is often used to compare the expression levels of different genes. In clinical testing, multiplex PCR can be used to detect two or more pa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/34C12Q1/68C12Q1/04
Inventor 徐定邦朱德芬陈有容徐文慧
Owner 徐定邦
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