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Typhoid fever salmonella and salmonella paratyphi fluorescent quantitative polymerase chain reaction (PCR) detection kit and application thereof

A technology of Salmonella typhi and detection kit, applied in the fields of biotechnology and medicine, can solve the problems of long detection period, complicated operation, low sensitivity, etc., and achieve the effects of accurate and reliable results, avoidance of cross-contamination, and rapid, accurate and sensitive results.

Active Publication Date: 2013-03-20
深圳生科原生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented assay allows salmolithiasis bacterium (SAL) from food animals such as fish or shrimp that causes enteritis caused by certain types of organisms like SARC strain O157:

Problems solved by technology

This patented technical problem addressed in this patent relates to identifying and distinguishing between various types of tickborne vims that cause symptoms like heat intolysema syndrome and encephalitis without causing false positives. Current tests involve testing all three kinds of organisms separately, making their identification process time-consuming and labor-demanding. Additionally, these techniques require special equipment and trained professionals, leading to potential issues related to reproducibility during implementation across medical centres.

Method used

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  • Typhoid fever salmonella and salmonella paratyphi fluorescent quantitative polymerase chain reaction (PCR) detection kit and application thereof
  • Typhoid fever salmonella and salmonella paratyphi fluorescent quantitative polymerase chain reaction (PCR) detection kit and application thereof
  • Typhoid fever salmonella and salmonella paratyphi fluorescent quantitative polymerase chain reaction (PCR) detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Preparation of fluorescent quantitative PCR detection kits for Salmonella typhi and Salmonella paratyphi A.

[0046] Prepare the first reaction solution of the present invention according to the following components in the PCR reaction tube:

[0047]

[0048] Add sterile ultrapure water to bring the total volume to 18.0 µl

[0049] Among them, the 5' end of the above-mentioned probe 117A (SEQ ID NO: 3) is connected with FAM, and the 3' end is connected with DABCYL; the 5' end of the above-mentioned probe 118H (SEQ ID NO: 6) is connected with HEX, and the 3' end Connected with DABCYL.

[0050] Mix the first reaction solution evenly, and then add 20 microliters of melted paraffin to the liquid surface. After it solidifies, the paraffin seals the first reaction solution below, and then adds the second reaction solution according to the following composition ratio:

[0051] DNA polymerase O (42U / μL) 0.2 μL

[0052] Bromophenol blue 0.01 μl

[0053] Then add sterilize...

Embodiment 2

[0055] Preparation of fluorescent quantitative PCR detection kits for Salmonella typhi and Salmonella paratyphi A.

[0056] Prepare the first reaction solution of the present invention according to the following components in the PCR reaction tube:

[0057]

[0058] Add sterile ultrapure water to bring the total volume to 18.0 µl.

[0059] Among them, the 5' end of the probe 117A (SEQ ID NO: 3) is connected to HEX, and the 3' end is connected to BHQ; the 5' end of the above probe 118H (SEQ ID NO: 6) is connected to FAM, and the 3' end There is BHQ connected.

[0060] Mix the first reaction solution evenly, and then add 20 microliters of melted paraffin to the liquid surface. After it solidifies, the paraffin seals the first reaction solution below, and then adds the second reaction solution according to the following composition ratio:

[0061] DNA polymerase O (42U / μL) 0.5 μL

[0062] Bromophenol blue 0.02 μl

[0063] Then add sterilized ultrapure water to make up to 2...

Embodiment 3

[0065] Application of fluorescent quantitative PCR detection kit for Salmonella typhi and Salmonella paratyphi A.

[0066] 1. Blood sample collection

[0067] For 20 cases of typhoid fever patients in the first week of aseptic collection of venous blood 5mL / person.

[0068] 2. Real-time fluorescent quantitative PCR detection

[0069] Take 5 μL of the venous blood collected in step 1 and directly add it to the PCR reaction tube of the PCR detection kit prepared in Example 1. The reaction tube contains the first reaction solution and the second reaction solution, which are separated by paraffin, and then covered Cover, put the PCR reaction tube into a fluorescent quantitative PCR instrument for PCR reaction. Wherein the PCR reaction conditions are:

[0070] 50℃: 2min;

[0071] 94°C: 2min;

[0072] 94°C: 5s, 55°C: 40s; 40 cycles.

[0073] After the reaction is finished, the reaction result is obtained.

[0074] 3. Result judgment

[0075] Judgment results based on the a...

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Abstract

The invention belongs to the field of biotechnology and medical science, and provides a typhoid fever salmonella and salmonella paratyphi fluorescent quantitative polymerase chain reaction (PCR) detection kit. The detection kit comprises a first reaction solution, a second reaction solution and paraffin, wherein the first reaction solution comprises a reaction buffer solution, a primer pair and an ecprobe aiming at typhoid fever salmonella, a primer pair and an ecprobe aiming at the salmonella paratyphi. The sequence of the primer pair aiming at the typhoid fever is SEQ ID NO: 1 and SEQ ID NO: 2, the sequence of the ecprobe aiming at the typhoid fever is SEQ ID NO: 3, the sequence of the primer pair aiming at the salmonella paratyphi is SEQ ID NO: 3 and SEQ ID NO: 3, and the sequence of the ecprobe aiming at the salmonella paratyphi is SEQ ID NO: 6. The second reaction solution comprises deoxyribonucleic acid (DNA) polymerase O. The invention further provides an application of the detection kit in detection of the typhoid fever salmonella and salmonella paratyphi. According to the detection kit, the detection time is shortened, cross contamination among samples is avoided, and the result is accurate and reliable.

Description

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Claims

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Application Information

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Owner 深圳生科原生物有限公司
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