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Oligonucleotide mixture, fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and application

An RT-PCR, oligonucleotide technology, applied in the field of pathogenic microorganism detection, can solve the problem of large missed detection risk, and achieve the effect of ensuring accuracy, preventing false positive results, and good inclusiveness

Pending Publication Date: 2022-05-13
深圳市赛格诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as the newly published nucleic acid sequences of HPIVs continue to increase, it can be seen through sequence comparison analysis that the primer probe sequences of existing HPIVs fluorescent RT-PCR detection reagents have different degrees of mismatching, which has a greater risk of missed detection

Method used

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  • Oligonucleotide mixture, fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and application
  • Oligonucleotide mixture, fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and application
  • Oligonucleotide mixture, fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: This embodiment provides a design scheme of fluorescent RT-PCR primers and probes for detecting HPIV1-3 and human RNase P nucleic acid.

[0053] The primers and probes involved in the present invention are designed and screened according to the following principles:

[0054] (1) The positions of primers and probes are selected in the conserved sequence region of the target genome to reduce missed detection (false negatives), and this region must be exclusive to other organisms to avoid false positives (non-specific amplification).

[0055] (2) The distance between the 5' end of the probe and its upstream primer is no more than 30 bp, preferably no more than 10 bp, so as to ensure efficient cutting of the probe by Taq DNA polymerase to generate a fluorescent signal.

[0056] (3) The GC content is 30-80%; the Tm value of the primer is about 58-60°C, and the Tm value of the probe is about 68-70°C. The Tm difference between the forward and reverse primers sho...

Embodiment 2

[0070] Example 2: This example demonstrates that the fluorescent RT-PCR detection reagent of the present invention is made into a freeze-dried reagent by vacuum freeze-drying technology.

[0071] Prepare fluorescent RT-PCR liquid detection reagents according to the ingredients and contents described in Table 2 (primers and probes in the ingredients are prepared according to Example 1), and the reagents are divided into 200 μl PCR eight-connected tubes according to 1 part / tube and then carried out Vacuum freeze-drying, the obtained lyophilized fluorescent RT-PCR detection reagent is white particles ( Figure 5 ). Add 23 μl of nuclease-free water to a reagent, shake slowly for 10 seconds, the reagent is completely dissolved, and the volume measured with a pipette is 25 μl, which shows that the volume of the dry matter of the reagent before reconstitution is equivalent to 2 μl. In order to prevent the reagents from getting wet and exposed, the reagents are first vacuum-treated w...

Embodiment 3

[0072] Example 3: This example demonstrates the detection effect of the freeze-dried fluorescent RT-PCR detection reagent of the present invention on HPIV1-3 and human RNase P nucleic acid positive reference products.

[0073]For HPIV1-3 and human RNase P RNA positive reference substance mixture, perform 10-fold serial dilution, mix 5 μl and 18 μl nuclease-free water for each concentration, and then add to 1 part of lyophilized fluorescent RT- In the PCR detection reagent; 23 μl of nuclease-free water was used as a negative control substance. A reaction mixture with a volume of 25 μl was obtained, which was dissolved by slow shaking, centrifuged, and tested on a fluorescent PCR instrument. The reaction program was set according to Table 3. Do 2 repetitions for each treatment.

[0074] Test results such as Image 6 As shown, the detection results of the lyophilized fluorescent RT-PCR detection reagent of the present invention are all positive to no less than 5 copies of HPIV1...

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Abstract

The invention discloses an oligonucleotide mixture, a fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and application. The oligonucleotide mixture contains one or more of the following oligonucleotide combinations A, B and C: the oligonucleotide combination A consists of three oligonucleotides, and the sequences of the oligonucleotides are respectively SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3; the oligonucleotide combination B consists of three oligonucleotides, and the sequences of the oligonucleotides are respectively SEQ ID No.4, 5 and 6; and the oligonucleotide combination C consists of four oligonucleotides, and the sequences of the oligonucleotides are respectively SEQ ID No.7, 8, 9 and 10. The oligonucleotide mixture disclosed by the invention is designed based on the latest HPIV1-3 nucleic acid sequence information and screened based on a specific screening principle, has good inclusiveness, specificity and reaction performance, and can ensure the detection accuracy.

Description

technical field [0001] The invention relates to pathogenic microorganism detection technology, in particular to an oligonucleotide mixture, a fluorescent RT-PCR detection reagent and applications. Background technique [0002] Human parainfluenza viruses (HPIVs) are one of the most common pathogens (others include rhinoviruses, coronaviruses, adenoviruses, etc.) that cause upper respiratory tract infections (such as the common cold and sore throat), and can also cause recurrent Lower respiratory tract diseases (such as pneumonia, bronchitis and bronchiolitis) are generally susceptible to people, especially children, the elderly and people with immunodeficiency. According to statistics, about 1 / 3 of acute respiratory infections in infants and young children are caused by HPIVs. HPIVs can be transmitted through contact with infectious pollutants or droplets of inhaled pollutants, and enter the human body from the eyes, mouth or nasal cavity, causing infection. [0003] HPIVs...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/166C12Q2521/531C12Q2563/107
Inventor 黎绍基林镜中朱碧莹刘钰涵
Owner 深圳市赛格诺生物科技有限公司
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