Kit for detecting human cytomegalovirus

A technology of human cytomegalovirus and cytomegalovirus, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of incomparable quantitative results, missed detection of clinical patients, and no traceability, etc., to achieve Suitable for popularization and application, high sensitivity and good specificity

Inactive Publication Date: 2018-12-18
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using real-time fluorescent PCR technology to detect HCMV DNA has the advantages of high sensitivity, good specificity, and easy operation. Although there are companies that have applied PCR technology to HCMV DNA detection, the quantitative units of each manufacturer have not been traced to the World Health Organization (WHO ) standard unit IU / mL, and the sensitivity is low (400 Copies / mL), resulting in missed detection of clinical patients, and the quantitative results of various manufacturers are not comparable and traceable

Method used

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  • Kit for detecting human cytomegalovirus
  • Kit for detecting human cytomegalovirus
  • Kit for detecting human cytomegalovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of a kit for detecting human cytomegalovirus

[0034] The composition of kit of the present invention:

[0035] (1) 20ul reaction solution 1: 50mM tris(hydroxymethyl)methylglycine (pH8.0), 100mM potassium acetate, 0.6uM primer 1, 0.6uM primer 2, 0.3uM primer 3, 0.3uM primer 4, 0.25uM Probe 1, 0.25uM Probe 2, 10U Taq enzyme, 0.5U UNG, 0.3mMdATP, 0.3mMdGTP, 0.3mMdCTP, 0.3mMdUTP, 0.1% sodium azide;

[0036] The sequences of 4 specific primers and 2 specific probes are:

[0037] Primer 1: 5'-AACTGCCTCACCCACCTGC-3' (SEQ ID No.1);

[0038] Primer 2: 5'-AACGATGGAGGACGACCAACAC-3' (SEQ ID No.2);

[0039] Primer 3: 5'-GAAGGCTCATGGCAAGAAAG-3' (SEQ ID No.3);

[0040] Primer 4: 5'-CTCACTCAGTGTGGCAAAGG-3' (SEQ ID No.4);

[0041] Probe 1: FAM- AAACACCATCTTTCCGGAGGTGCGGT-BHQ1 (SEQ ID No. 5);

[0042] Probe 2: ROX-CTTGAGGTTGTCCAGGTGAGCCAG-BHQ2 (SEQ ID No. 6).

[0043] (2) 10ul reaction solution 2: including 1.5mM Mn(OAc) 2 and 0.1% sodium azide.

[0044] (...

Embodiment 2

[0050] Example 2 Human cytomegalovirus nucleic acid extraction and amplification method

[0051] The operation steps of using the kit prepared in Example 1 to detect human giant cell DNA in clinical urine samples are:

[0052] (1) Sample extraction

[0053] 1. Take 1ml of urine and add an equal volume of sample concentrate D, vortex evenly, centrifuge at 12000rpm for 5min, discard the supernatant; add 600µl normal saline, vortex mix, and set aside.

[0054] 2. At the same time, take 600 μl negative quality control, strong positive quality control, weak positive quality control, standard A, B, C, D and the sample to be tested, add 20 μl DNA internal standard respectively, cooperate with Zhengzhou Antu Bioengineering Co., Ltd. magnetic bead method nucleic acid extraction reagent to extract nucleic acid DNA in each sample and set aside.

[0055] (2) Reagent preparation

[0056] According to the number of samples to be tested, negative quality control, strong positive quality c...

Embodiment 3

[0066] Embodiment 3 Sensitivity experiment of the kit of the present invention

[0067] Use negative urine to dilute HCMV-positive clinical samples (quantity can be traced to WHO) to 34.5IU, use the kit prepared in Example 1 to detect 20 samples, and the obtained amplification curve is as follows figure 1 shown. From figure 1 It can be seen that the kit of the present invention detects 20 positive clinical samples of 34.5IU / mL, and all 20 are detected, which proves that the detection limit of the kit of the present invention can reach 34.5IU / mL, and its sensitivity is very high.

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Abstract

The invention discloses a kit for detecting human cytomegalovirus. The kit comprises specific PCR primers and a fluorescent probe for detecting human cytomegalovirus DNAs, specific primers and a probefor detecting a DNA internal standard, a PCR reaction buffer, a mixed enzyme liquid, theDNA internal standard, a negative control, a strongly positive control, a weakly positive control and quantitative references A, B, C and D; wherein the sequences of the primers and the probes are as shown in SEQ ID No. 1-6. The kit of the invention has the advantages of convenient and quick detection, capacity of quantitatively detecting human cytomegalovirus within 20 min, high sensitivity, the lowest detection limit of 34.5 IU/ml, and good specificity. According to the invention, a pair of primers are designed according to the internal repetitive sequence [mu]l65, so the primers have no cross-interference with other pathogens and have high specificity. The kit of the invention can be traced to the World Health Organization (WHO) standard unit IU/mL; quantitative results have traceability; and the kit has high reference value for clinical diagnosis of diseases related to human cytomegalovirus andis suitable for promotion and application.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a kit for detecting human cytomegalovirus. Background technique [0002] Human cytomegalovirus (HCMV) belongs to the β-subfamily of the Herpesviridae family and contains linear double-stranded DNA molecules with a length of 220-240kb. It is similar to other herpesviruses in shape and genetic structure. HCMV is the most common virus causing congenital infection worldwide, with an infection rate of 0.2%-2.0% in live births, twice that of rubella virus infection. Its infected population spreads all over the world, and most people acquire the infection in childhood or youth. With the increase of age, the positive rate of antibodies also increases, and there is no significant difference between men and women, suggesting that HCMV infection is very common and often causes multiple infections. Systemic diseases, fetal liver involvement is more common, in addition, it can also invade...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/701C12Q2600/166C12Q2545/101
Inventor 李静静李振红杜美鲁清月付光宇吴学炜苗拥军
Owner AUTOBIO DIAGNOSTICS CO LTD
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