Treponema pallidum real-time fluorescence quantitative PCR (Polymerase Chain Reaction) multitarget detection kit and preparation thereof

A real-time fluorescence quantitative, Treponema pallidum technology, applied in the field of kits, can solve the problems of low accuracy, insufficient sensitivity and specificity, and achieve the effect of avoiding missed detection, improving accuracy and sensitivity, and improving sensitivity

Active Publication Date: 2011-11-23
ZHONGSHAN HOSPITAL XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

What's more serious is that the sensitivity and specificity of the current PCR detection reagents are seriously insufficient d

Method used

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  • Treponema pallidum real-time fluorescence quantitative PCR (Polymerase Chain Reaction) multitarget detection kit and preparation thereof
  • Treponema pallidum real-time fluorescence quantitative PCR (Polymerase Chain Reaction) multitarget detection kit and preparation thereof
  • Treponema pallidum real-time fluorescence quantitative PCR (Polymerase Chain Reaction) multitarget detection kit and preparation thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0047] The preparation method of the real-time fluorescent quantitative PCR multi-target detection kit of Treponema pallidum comprises the following steps:

[0048] 1) Target gene screening

[0049] Screening of high-specificity and high-sensitivity target genes of Treponema pallidum from GENBANK. Five highly specific syphilis genes including polA gene, TPN47 gene, tmpA gene, TpF-1 gene and bmp gene. The design of primers and fluorescent probes was assisted by primer premier 5.0, Oligo 6.0, TM Utility vl.3, Clustal X and other design software, and then homology comparison was performed by BLAST to ensure their specificity. The synthesized primers and probes were dissolved with TE (10mM Tri-HCl, pH 5.0, 0.1mM TA), and the concentrations were determined using an ND-1000UV-VIS wavelength ultraviolet / visible light scanning spectrophotometer (Nanedrop, USA). The concentration was adjusted to 50 μM and stored at -20°C.

[0050] 2) Construction of multi-target gene standard plasmi...

Embodiment 1

[0084] 1) Target gene screening

[0085] References, Screening Treponema pallidum High Specificity and High Sensitivity Target Genes from GENBANK. Five highly specific syphilis genes including polA gene, TPN47 gene, tmpA gene, TpF-1 gene and bmp gene. The design of primers and fluorescent probes was assisted by primer premier 5.0, Oligo 6.0, TM Utility vl.3, Clustal X and other design software, and then homology comparison was performed by BLAST to ensure their specificity. The synthesized primers and probes were dissolved with TE (10mMTri-HCl, pH5.0, 0.1mM TA), and the concentrations were determined using an ND-1000UV-VIS wavelength ultraviolet / visible light scanning spectrophotometer (Nanedrop, U.S.), and the final concentration Adjust to 50μM and store at -20°C.

[0086] 2) Construction of multi-target gene standard plasmid

[0087] The DNA of Treponema pallidum Nichols strain was extracted by the silica gel membrane-spin column method as a template, and the primers of t...

Embodiment 2

[0109]Similar to Example 1, the difference is that the samples to be tested are tissue samples of chancre and skin lesions, and the samples are washed with an appropriate amount of sterilized physiological saline to wash the blood stained with the tissue for inspection; take about 50 mg of tissue, add 1 mL of sterilized physiological saline for use Grind it into tissue homogenate with a homogenizer, transfer to a 1.5mL centrifuge tube, centrifuge at 12,000rpm for 5min; remove the supernatant, add 1mL of sterilized saline to the precipitate and mix well, and centrifuge at 12,000rpm for 5min; remove the supernatant, extract DNA from the precipitate and PCR amplification is the same as in Example 1.

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Abstract

The invention relates to a kit, particularly a Treponema pallidum real-time fluorescence quantitative PCR (Polymerase Chain Reaction) multitarget detection kit and preparation thereof. The kit comprises a DNA (deoxyribonucleic acid) extraction reagent bottle, a heat-resistant DNA polymerase bottle, a PCR reaction liquid bottle, a standard substance bottle, a quality control substance bottle and apacking box, wherein the DNA extraction reagent bottle comprises a protease K bottle, a lysis buffer bottle, an anhydrous alcohol bottle, an inhibitor removing liquid bottle, a first cleaning buffer bottle, an eluent bottle, a second cleaning buffer bottle and a centrifugation column containing a collection tube; the standard substance bottle is provided with 6 standard substance plasmid bottles containing 5 target genes; and the quality control substance bottle comprises a negative quality control substance bottle and a positive quality control substance bottle. The preparation method comprises the following steps: screening the target genes, and constructing the multitarget gene standard substance plasmids; after making a multitarget gene detection reagent standard curve, sequentially preparing PCR reaction liquid and heat-resistant DNA polymerase; after establishing a sample processing method, preparing the quality control substance; and finally, completing the Treponema pallidum real-time fluorescence quantitative PCR multitarget detection kit.

Description

technical field [0001] The invention relates to a kit, in particular to a treponema pallidum real-time fluorescent quantitative PCR multi-target detection kit and a preparation method thereof. Background technique [0002] Syphilis (Syphilis) is a sexually transmitted disease caused by Treponema pallidum (TP), and its pathogen is Treponema pallidum, which belongs to the family Treponemaceae. Treponema pallidum is mainly transmitted through sexual contact, blood transfusion, wound or placenta. Treponema pallidum enters the blood from the lymph nodes near the infected area and spreads throughout the body, affecting almost all tissues and organs of the body. The clinical manifestation is systemic and can be divided into different clinical stages, including the first, second, third and latent stages. The World Health Organization (WHO) had optimistically predicted ([1] WHO. Global prevalence and incidence of selected curable sexually transmitted infections: Overview and estimat...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/01
Inventor 杨天赐张忠英林丽蓉刘莉莉方赞熙黄松洁张长弓
Owner ZHONGSHAN HOSPITAL XIAMEN UNIV
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