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Freeze-dried Mycobacterium Tuberculosis Nucleic Acid Detection Kit(Enzymatic Probe Isothermal Amplification)

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Freeze-dried Mycobacterium Tuberculosis Nucleic Acid Detection Kit(Enzymatic Probe Isothermal Amplification)

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Introduction

[Product Name]
Freeze-dried Mycobacterium Tuberculosis Nucleic Acid Detection Kit(Enzymatic Probe Isothermal Amplification)
[Packaging Size] 48 tests/kit
[Intended Use]
This kit is used for in vitro qualitative detection of tuberculosis nucleic acid in sputum samples of suspected patients infected with mycobacterium tuberculosis with tuberculosis-related signs/symptoms and X-ray examination results, and other persons who need to be diagnosed or differentially diagnosed with mycobacterium tuberculosis infection.
Mycobacterium culosis, referred to as Tubercle bacillus(TB), is a class of obligate aerobic bacteria with positive acid-fast staining. It is amastigote and has fimbriae and microcapsules but not forms spores. Its bacterial wall has neither teichoic acid of gram-positive bacteria, nor lipopolysaccharide of gram-negative bacteria. Mycobacterium tuberculosis, which is pathogenic to humans, is generally considered to be human, bovine, and African. Its pathogenicity may be related to the inflammation caused by the proliferation of bacteria in tissue cells, the toxicity of bacterial components and metabolites, and the immune damage to the bacterial components. Pathogenic substances are associated with capsules, lipids and proteins. Mycobacterium tuberculosis can invade susceptible organisms through the respiratory tract, digestive tract or skin injury, causing tuberculosis of various tissues and organs, of which the most common is pulmonary tuberculosis through the respiratory tract. It usually occurs in children, and presents with symptoms such as low-grade fever, night sweats, and a small amount of hemoptysis. Secondary infection is mainly manifested as low-grade fever, night sweats, and hemoptysis. Mostly it is long-term chronic disease, and a few are acute. Tuberculosis is one of the top ten causes of human death worldwide. In 2018, about 10 million people around the world were infected with Mycobacterium tuberculosis, of which about 1.6 million died.
IS6110 is an inserted nucleotide sequence discovered by Thierry et al. in 1990 in Mycobacterium tuberculosis. IS6110 is present in multiple copies of a sequence in the Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, and BCG genes. IS6110 is relatively stable in the strains in which it exists, and will not change the IS6110 DNA fingerprints when the strains develop drug-resistant mutations. The 38KD protein antigen is a phosphate transporter and contains 6 epitopes targeted by monoclonal antibodies specific for Mycobacterium tuberculosis. The diagnostic specificity of the reported 38KD is 88%-100%.

Experiment operators should have received professional training in gene amplification or molecular biology method detection, have relevant experimental operation qualifications, and laboratories should have corresponding biosafety protection conditions. The test results of the kits are not the only indicators for the diagnosis of mycobacterium tuberculosis infection in patients, and must be comprehensively analyzed in combination with the clinical manifestations of patients and other test results.

[Test Principles]

This kit adopts Enzymatic Probe Isothermal Amplification (EPIA), and designs specific primers and RNA base-containing probes (rProbe) for the highly conserved region of mycobacterium tuberculosis(IS6110 and 38KD), and adds Bst enzyme and RNaseH enzyme at the same time, in which the left and right ends of the RNA base of rProbe are labeled with fluorescent groups and quencher, respectively. Use the DNA polymerase activity and strand displacement activity of Bst enzyme to amplify the target to be tested at a constant temperature, the RNaseH enzyme can cleave the RNA bases on the target-probe hybrid chain, so that the fluorescent group and quencher of rProbe are separated thereby fluorescing. In addition, the left fragment of the residual rProbe RNA base can be used as a primer to further extend to form a product, which further accumulates the product. The fluorescent signal is continuously accumulated with the formation of the product, thereby realizing the detection of the target nucleic acid.

In the system, the recombinant pine chloroplast gene plasmid is used as an exogenous internal reference to control the quality of the reagents and the operation itself to avoid false negatives.

[Storage Conditions and Shelf-life]

1. Unopened kit: It should be stored at no higher than 30°C and protected from light, and the shelf-life is 12 months.
2.Test should be performed immediately after reconstitution of the freeze-dried positive control and internal control. The remaining positive control and internal control should be stored below -15°C and protected from light. The validity period is 1 week, and repeated freezing and thawing should be avoided ( limited to 2 cycles).

3.Transportation conditions: It can be transported stably for 1 month when the temperature is not higher than 37°C.
4. See the packaging label for the production date, production batch number and expiration date.

[Applicable Instruments]

Applied Biosystems 7500 Real-Time PCR Systems, SLAN-96P Real-Time PCR Systems(Shanghai Hongshi Medical Technology Co., Ltd.), LightCycler®480 Real-Time PCR system, Real-time Fluorescence Constant Temperature Detection System Easy Amp HWTS1600.

[Requirements for Samples]

1. Sample type: sputum.
2. Sample collection: For those with less sputum, let the patient rinse his mouth after waking up in the morning, then instruct or assist him/her to take a deep cough, cough up the sputum from the lungs, and collect it in a clean container for testing as soon as possible. For those with much sputum, first ask the patient to cough lightly and cough out the old sputum retained in the trachea, and then instruct or assist him/her to take a deep cough to collect fresh sputum from the depths of the lungs, and send it for testing immediately. If there is no sputum, you can go to the hospital for atomization inhalation to induce sputum, and then send it for testing.
3. Precautions for sample collection: Avoid contamination during sample collection, storage and transfer. It is forbidden to use external respiratory medicines (ointments, sprays, drops) within 24 hours before sample collection;
All samples are considered to be potentially infectious, and operations are performed

in accordance with relevant national standards.
4.Storage: The sputum samples to be tested could be stored at 2−8°C for 24 hours; stored below -15°C for no more than 6 months; can be stored -70°C for long time. The extraction of frozen samples should be performed after the samples are completely thawed, and repeated freezing and thawing should be avoided. If the extracted DNA sample is not used for immediate detection, it should be placed below -15°C, the storage period should not exceed 6 months, and the number of repeated freezing and thawing should not exceed 4 cycles.

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