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Kit for detecting active pulmonary tuberculosis

A tuberculosis and active technology, applied in the field of kits for detecting active tuberculosis, can solve the problems of unreliability, lack of tuberculosis-specific serum miRNAs composition research reports, and low specificity, achieving high accuracy and design Accurate, early warning and early diagnosis effect

Active Publication Date: 2013-04-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the requirements of disease biomarker research, 1-2 miRNAs are used as disease markers, with low specificity
At the same time, due to the individual differences of different patients, it is extremely unreliable to use only 1-2 miRNAs to judge the disease
However, there is no research report on the composition of tuberculosis-specific serum miRNAs

Method used

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  • Kit for detecting active pulmonary tuberculosis
  • Kit for detecting active pulmonary tuberculosis
  • Kit for detecting active pulmonary tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A diagnostic kit for detecting active pulmonary tuberculosis, the kit has RNA extraction buffer, active pulmonary tuberculosis specific serum miRNA composition: hsa-miR-29c, hsa-miR-22, hsa-miR-320b and hsa-miR-101 and internal reference reverse transcription primers and PCR primers, fluorescent quantitative RT-PCR reaction solution.

[0032] The reverse transcription primer sequence is:

[0033]hsa-miR-29c:5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTAACCG-3';hsa-miR-22:5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGTT-3';hsa-miR-320b:5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCCGTT-3';hsa-miR-101:5' -GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTCAGT-3';

[0034] Internal reference hsa-miR-16: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCGCCAA-3'.

[0035] The PCR upstream primer sequence is:

[0036] hsa-miR-29c: 5'-GGCGGTAGCACCATTTGAA-3';

[0037] hsa-miR-22: 5'-CGGAAGCTGCCAGTTGAAGA-3';

[0038] hsa-miR-320b: 5'-AAAAGCTGGGTTGAGAGGGCA-3';

[0039...

Embodiment 2

[0047] Example 2 Kit application

[0048] Detect the expression levels of hsa-miR-29c, hsa-miR-22, hsa-miR-320b and hsa-miR-101 in the serum of patients with active pulmonary tuberculosis, healthy controls, lung cancer patients, pneumonia patients and patients with chronic obstructive pulmonary disease .

[0049] Sample collection: 82 patients with active pulmonary tuberculosis, 108 healthy controls, 30 patients each with lung cancer, pneumonia and chronic obstructive pulmonary disease.

[0050] Collect the total RNA in the serum sample: add 700 μL Qiagen lysate to 200 μL serum sample and mix well, let stand for 5 minutes; add 140 μL chloroform, shake vigorously, let stand for 3 minutes; 12000 rpm, 4 ℃ for 15 minutes and take For the supernatant, add 1.5 times the volume of absolute ethanol and mix well; add the above mixture to the column, centrifuge at 8000 rpm for 18 seconds, discard the collected solution; add 700 μL RWT solution, centrifuge at 8000 rpm for 18 seconds, di...

Embodiment 3

[0066] Example 3 Establishment of specific serum hsa-miR-29c, hsa-miR-22, hsa-miR-320b and hsa-miR-101 compositions for active pulmonary tuberculosis

[0067]Using MedCalc software to calculate the ROC curve of single serum hsa-miR-29c, hsa-miR-22, hsa-miR-320b and hsa-miR-101 in the diagnosis of active pulmonary tuberculosis, including sensitivity, specificity and area under the curve (AUC) , the results showed that the AUCs of serum hsa-miR-29c, hsa-miR-22, hsa-miR-320b and hsa-miR-101 were 0.723, 0.711, 0.702, 0.706, and the sensitivities were 51.2%, 67.5%, 94.9% respectively %, 90.1%, and the specificities were 87.4%, 70.5, 36.4%, and 44.2%, respectively. The results showed that the sensitivity and specificity of a single serum miRNA in the diagnosis of active pulmonary tuberculosis were not high.

[0068] Using the Logistic stepwise regression model to calculate the ROC curve of serum hsa-miR-29c, hsa-miR-22, hsa-miR-320b and hsa-miR-101 composition in the diagnosis of a...

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Abstract

The invention provides a diagnostic kit for detecting active pulmonary tuberculosis. The kit consists of an RNA extract buffer solution, a specific serum miRNA composition for the active pulmonary tuberculosis, internal reference reverse transcription primers, polymerase chain reaction (PCR) primers and a fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) reaction solution, wherein the specific serum miRNA composition for the active pulmonary tuberculosis consists of four differential expression serum miRNAs, namely hsa-miR-29c, hsa-miR-22, hsa-miR-320b and hsa-miR-101. By using the specific serum miRNAs composition for the active pulmonary tuberculosis to detect the active pulmonary tuberculosis on the serum miRNAs level, the sensitivity is 90.2 percent, and the specificity is 75.0 percent; and the kit has early-stage diagnostic value for the active pulmonary tuberculosis, and can realize early warning and early diagnosis of the active pulmonary tuberculosis.

Description

technical field [0001] The invention belongs to the research of serum biological markers, and relates to a kit for detecting miRNA specific to active pulmonary tuberculosis. A specific miRNA capable of identifying active pulmonary tuberculosis is used to capture serum biological markers and analyzed by miRNA with quantitative control , to detect active pulmonary tuberculosis in serum. Background technique [0002] Tuberculosis is a chronic pulmonary infectious disease caused by Mycobacterium tuberculosis, which is a serious threat to human health. The epidemic situation in China is very severe; there are about 1.3 million active tuberculosis patients, 1.45 million new cases per year, accounting for 18% of the global population, and 130,000 deaths per year, exceeding The sum of deaths from other infectious diseases. The high morbidity and mortality of tuberculosis, as well as airborne transmission, imposes a considerable burden on the patient, his contacts, and society. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/32
Inventor 李继承张星
Owner ZHEJIANG UNIV
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