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Detection kit for ureaplasma urealyticum (UU) nucleic acid by utilizing RNA constant-temperature amplification

Ureaplasma urealyticum and detection kit technology, applied in the field of Ureaplasma urealyticum nucleic acid detection kits, can solve the problems of affecting PCR sensitivity, positive results in patient test results, false positive contamination, etc., and achieve high sensitivity , high specificity, low pollution effect

Active Publication Date: 2011-09-21
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there may be inhibitors of amplification in urine, which may affect the sensitivity of PCR
In addition to the fact that inhibitors in urine will affect the sensitivity of PCR and lead to false negative results, another disadvantage of PCR is that it is easy to cause false positive contamination, and the test results of patients who have recovered after medication still show positive results

Method used

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  • Detection kit for ureaplasma urealyticum (UU) nucleic acid by utilizing RNA constant-temperature amplification
  • Detection kit for ureaplasma urealyticum (UU) nucleic acid by utilizing RNA constant-temperature amplification
  • Detection kit for ureaplasma urealyticum (UU) nucleic acid by utilizing RNA constant-temperature amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Extraction of UU Nucleic Acid RNA

[0034] The specific steps of nucleic acid extraction are:

[0035] Preparation of positive control substance: Take 200 μl of normal saline, add 10 μl of positive control, mix well and set aside

[0036] Preparation of negative control substance: Take 200 μl of normal saline, add 10 μl of negative control, mix well and set aside.

[0037] Add 100 μl nucleic acid extraction solution, 200 μl urine sample preservation solution, 200 μl urine sample or swab washing solution or positive control or negative control substance to the sample processing tube (1.5ml centrifuge tube), change a tip for each sample added, vortex Rotate and mix; keep warm at 60°C for 5 minutes; place at room temperature for 10 minutes;

[0038] Place the sample processing tube on the magnetic bead separation device and let it stand for 5 minutes (if some magnetic beads are difficult to adsorb to the tube wall during the magnetic bead adsorption process, the adsorptio...

Embodiment 2

[0044] SAT nucleic acid amplification detection

[0045] Take 30 μl of the amplification detection solution (including magnetic beads) from the above amplification detection solution after shaking and mixing to a clean micro-reaction tube, incubate at 60°C for 10 minutes, and at 42°C for 5 minutes; at the same time, preheat the SAT enzyme solution to 42°C;

[0046] Add 10 μl of preheated enzyme solution to the micro-reaction tube (do not touch the micro-reaction tube with the sampling tip, and replace the tip if there is any contact), cover the tube after adding enzyme and shake at 1200rpm for 15 seconds to mix well;

[0047] Quickly transfer the micro reaction tube to a suitable constant temperature fluorescence detection instrument, react at 42°C for 40 minutes, set the fluorescence to be detected every 1 minute, and detect 40 times in total;

[0048] After the reaction is over, take out the micro reaction tube and soak it in 10% 84 disinfectant solution. Take the micro rea...

Embodiment 3

[0058] Preparation and assembly of components of the UU kit

[0059] The kits are divided into box A (specimen processing unit) stored at 2-30°C and box B (nucleic acid amplification detection unit) stored at -15-35°C.

[0060] Box A (specimen processing unit) consists of:

[0061] Urine sample preservation solution: containing 0.1-10M ammonium sulfate; 0.15-1M HEPES;

[0062] Nucleic acid extraction solution: containing magnetic beads 0.1-10mg / ml, TCO 0.1-100μM;

[0063] Washing solution: containing SDS 0.1-1%, NaCl 10-150mM, ethanol 0.1-1%;

[0064] Box B (nucleic acid amplification detection unit) consists of:

[0065] UU reaction solution: containing dNTPs and NTPs, mainly including Tris 10-50mM, MgCl2 10-40mM, dNTP0.5-5mM, NTP 1-10mM, PVP40 1-10%, KCl 5-40mM;

[0066] SAT enzyme solution: containing T7 RNA polymerase 200~2000U / reaction, M-MLV reverse transcriptase 400~4000U / reaction, 2~10mM HEPES pH7.5, 10~100mM N-acetyl-L-cysteine, 0.04~0.4 mM zinc acetate, 10~100mM...

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Abstract

The invention relates to a detection kit for ureaplasma urealyticum (UU) nucleic acid by utilizing RNA constant-temperature amplification. The detection kit comprises a urine sample preserving solution, a nucleic acid extraction solution, a UU reaction solution, a UU detection solution, an SAT (Spermidine / Spermine N1-Acetyltransferase) enzyme solution, a UU positive control and a UU negative control. The detection kit provided by the invention has the analytical sensitivity of 50 CFU / reaction for detecting UU, and has the characteristics of high specificity and high sensitivity; and an amplification product RNA is easy to degrade in a natural environment and causes low pollution.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnostic reagents, in particular to a nucleic acid detection kit for Ureaplasma urealyticum using RNA constant temperature amplification. Background technique [0002] Ureaplasma urealyticum (UU) is a prokaryotic microorganism with a size between bacteria and viruses. It is a common pathogen in the human genitourinary tract. It is related to infertility and is one of the pathogens of sexually transmitted diseases. It is worth noting that there are many pregnant women with Ureaplasma urealyticum infection, 80% reported abroad, and 55.12% reported in my country. Infection with Ureaplasma urealyticum during this period not only endangers the health of the mother, but also endangers the survival of the fetus. Serious consequences such as low birth weight infants, neonatal respiratory tract infections, central nervous system infections, and fetal death are prone to occur. [0003] After infection ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
Inventor 于明辉方亮张常娥居金良
Owner SHANGHAI RENDU BIOTECH
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