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Enterovirus nucleic acid detection kit (enzyme digestion probe constant temperature amplification method)

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Enterovirus nucleic acid detection kit (enzyme digestion probe constant temperature amplification method)

Hand, foot and mouth disease (HFMD) is mainly an acute infectious disease caused by enterovirus infection[1]. It mostly occurs in children under 5 years of age and can cause herpes on the hands, feet, and mouth.
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Introduction

【product name】

Generic name: Enterovirus nucleic acid detection kit (constant temperature amplification method of enzyme digestion probe)

[Packing specifications] 50 servings/box

【expected usage】

This kit is used to qualitatively detect enterovirus nucleic acid in human throat swabs in vitro.

Hand, foot and mouth disease (HFMD) is mainly an acute infectious disease caused by enterovirus infection[1]. It mostly occurs in children under 5 years of age and can cause herpes on the hands, feet, and mouth. A few After the onset of the patient, the nervous system is quickly involved, manifested as brainstem encephalitis, encephalomyelitis, cerebrospinal meningitis, etc., and the mortality rate of children who develop circulatory failure and neurogenic pulmonary edema is high [2]. There are more than 20 types of enteroviruses that cause hand, foot and mouth disease, including types 16, 4, 5, 9, and 10 of Coxsackie virus A group, types 2, 5 and enterovirus 71 of group B. It is a common pathogen of hand, foot and mouth disease, among which Coxsackie virus A16 (Cox A16) and enterovirus 71 (EV 71) are the most common [3-4].

This kit provides an auxiliary means for the diagnosis of hand-foot-mouth disease patients. The test results are for clinical reference only, and the final diagnosis should be considered in close conjunction with other clinical indicators.

【Principle of Detection】

This kit adopts Enzymatic Probe Isothermal Amplification (EPIA) technology, design specific primers and RNA base-containing probes (rProbe) for the highly conserved regions of EV, and add Bst enzyme, RNaseH and Reverse transcriptase, etc., where the left and right ends of the RNA base of rProbe are respectively labeled with a fluorescent group and a quencher group. First, the extracted RNA is reverse transcribed into cDNA, and the target is amplified by the DNA polymerase activity and strand displacement activity of Bst enzyme. RNaseH enzyme can cleave the RNA bases on the target-probe hybrid chain to make rProbe fluorescent group Separate from the quenching group to emit fluorescence. In addition, the left fragment of the remaining rProbe RNA base can be used as a primer to continue to extend to form a product, which further accumulates the product. The fluorescent signal accumulates with the formation of the product, thereby realizing the detection of the target nucleic acid.

【Main ingredients】

serial number

Composition(50人份/盒)

Specification

quantity

Composition description

1

EV nucleic acid reaction solution

1mL/支

1支

Constant temperature amplification buffer, dNTP, water, primer probe and RNaseH, etc.

2

EV detection enzyme solution

40μL/支

1支

Constant temperature amplification of Bst enzyme and reverse transcriptase, etc.

3

EV positive control substance

600µL/支

1支

106Copies/mL Target gene pseudovirus solution

4

EV negative control substance

600µL/支

1支

RNase and DNase-free water

Note: The components of products of different batch numbers cannot be mixed or interchanged.

Need to bring your own reagents: Viral RNA Extraction Kit (DP315-R) from Tiangen Biochemical Technology (Beijing) Co., Ltd., or Nucleic Acid Extraction Reagent (HWTS-3001) from Jiangsu Hongwei Tesi Medical Technology Co., Ltd.

Self-provided test materials: 1.5mL centrifuge tube without RNase and DNase, RNase and DNase Tip-free, bench-top centrifuge, bench-top shaking mixer.

[Storage conditions and validity period]

This kit is stored below -18°C, and the kit is valid for 9 months. The number of repeated freezing and thawing is not more than 4 times, and it can be stored stably for 5 days when it is transported under the dark condition below -18℃.

The production date and expiry date are shown on the packaging label.

【Applicable equipment】

ABI 7500 fluorescence quantitative PCR instrument, Shanghai Hongshi SLAN-96P fluorescence quantitative PCR instrument

【Sample requirements】

sample collection

Freshly collected throat swab.

Pharyngeal swab sample collection: Use a polyester fiber head swab to wipe the posterior pharyngeal wall and bilateral tonsils of the uvula (palatine or uvula) with moderate force several times to avoid touching the tongue. Take out the posterior sampling tube and break it. Place the plastic handle on the hand contact area so that the swab is immersed in the sampling solution, and the tube cap is tightened.

save

The sample to be tested should be stored at 2~8℃ for no more than 6 days, below -18℃ for no more than 3 months, and samples below -70℃ should be stored for a long time. Repeated freezing and thawing of samples should be avoided, and the number of repeated freezing and thawing should not exceed 4 times.

transportation

Using foam box and dry ice seal for transportation, the transportation time is no more than 5 days.

【Testing method】

Amplification reagent preparation

Take out the EV nucleic acid reaction solution and the EV detection enzyme solution from the kit, wait for them to dissolve, shake and mix, and centrifuge briefly. According to the required number of samples n, calculate the number of samples to be prepared n (n=number of samples + negative control product + positive photo product), prepare amplification reagents according to the following table, mix well and centrifuge for a short time, and distribute them into eight-way reaction tubes. The used EV nucleic acid reaction solution and EV detection enzyme solution should be frozen and stored below -18°C immediately.

Element

EV nucleic acid reaction solution

EV detection enzyme solution

Amplification reagent ratio

19.3μL×n

0.7μL×n

 
Sample processing
 
Take 1.5 mL RNase and DNase-free centrifuge tubes, label them as negative control substance, test sample, and positive control substance in sequence. Follow the instructions of the recommended nucleic acid extraction reagent for subsequent sample RNA extraction. (Positive control substance and negative control substance are required Extract the same amount as the sample at the same time).
 
The extracted RNA samples should be used for testing immediately or stored below -70°C.
 
Loading
 
Add 10μL each of the RNA of the sample to be tested processed in step 2 and 10μL each of the negative control substance and the positive control substance into each set reaction tube, tightly cap the tube, and centrifuge briefly.
 
Real-time constant temperature amplification
 
Instrument channel and reaction volume selection
 
Select FAM channel (Reporter: FAM, Quencher: None) to detect enterovirus nucleic acid;
 
The reaction volume (Sample Volume) is 30µL. For specific detection channel settings, please refer to the instructions for each instrument.
 
Real-time constant temperature amplification condition setting
 

Step

temperature

time

Number of cycles

Collect fluorescent signal

1

50℃

15min

1

no

2

60℃

1min

30

Yes

 

Result analysis

Tt value: the abscissa reading of the intersection of the amplification curve and the threshold line in each reaction tube.

ABI7500 fluorescence quantitative PCR instrument baseline and threshold setting method

Baseline setting: Baseline setting: Use the default baseline. If the minimum Tt value in the sample is less than the default baseline end point, it needs to be adjusted according to the actual results, as follows: When setting a channel baseline, first select the minimum Tt Value of the sample to be tested, change the option "☑Auto Baseline" to "□Auto Baseline", then select ☑Baseline Start, and manually adjust the baseline end point to be less than the minimum Tt value of the sample. .

Threshold setting: When setting the threshold line of a certain channel, first select the negative control for detection, remove the checked automatic threshold line, change the option "☑Auto line" to "□Auto", and then manually adjust the threshold line. The FAM channel is based on the threshold line just exceeding the highest point of the normal negative control amplification curve (irregular noise line).

Shanghai Hongshi SLAN-96P fluorescent quantitative PCR instrument baseline and threshold setting method

baseline (baseline) setting: set the baseline start point and end point in "Create Project" → "Experiment Parameters" to 1. If the minimum Tt value in the sample to be tested is less than 2, it needs to be adjusted according to the actual results. The details are as follows: when setting the baseline of a certain channel, adjust the "baseline end point" in the "basic parameters" to be less than the Tt value.

Threshold setting: When setting the threshold line of a certain channel, first set the "baseline optimization" in the "basic parameters" to manual optimization, and then manually adjust the threshold line so that the threshold line just exceeds the normal negative control for amplification The highest point of the curve (irregular noise line) shall prevail.

QC:

Negative control substance: There should be no obvious amplification curve or no value.

Positive control: There should be an obvious amplification curve and the Tt value should not be greater than 28.

The above requirements must be met at the same time in the same experiment, otherwise, this experiment is invalid and needs to be repeated.

【Positive judgment value or reference interval】

The ROC curve analysis method is used to analyze the critical value of the kit. It is determined that the positive judgment value of this kit for detecting enterovirus is Tt value <30.

【Interpretation of test results】

If the test sample has an obvious amplification curve and the Tt value is less than 30, it is judged as positive for enterovirus.

If the test sample has no amplification signal, it is judged that the enterovirus test is negative.

【Limitations of the test method】

The test results of this kit are for clinical reference only, and the clinical diagnosis and treatment of patients should be comprehensively considered in conjunction with their symptoms/signs, medical history, other laboratory tests, and treatment responses.

Unreasonable sample collection, transportation, storage and processing procedures may lead to erroneous test results.

Contamination of amplification products and cross-contamination between samples in nucleic acid extraction are prone to false positive results. Therefore, clinical laboratories should strictly follow the "Clinical Gene Amplification Laboratory Work Specifications" with equipment and operators, and should strictly follow the instructions operate.

A negative test does not mean that the patient is a non-enteric virus infection patient. The specific diagnosis result must be judged in conjunction with other clinical diagnosis results. The negative test may be caused by: ①Unreasonable sample collection, transportation and processing, low nucleic acid content in the sample, resulting in test failure or inaccurate results; ②The detected target sequence region changes or sequence changes caused by other reasons may cause Inaccurate results; ③Other interference factors or nucleic acid reaction inhibitors that have not been verified may also cause inaccurate results.

【Product performance index】

The appearance of the kit is intact, and the liquid components are clear, transparent and free of insoluble matter.

The negative control product tested by the kit should have no obvious amplification curve or no value; the positive control product tested should have a clear amplification curve and the Tt value should not be greater than 28.

The kits tested the positive reference products P1-P4 of the enterprise were all positive.

The kits tested the negative reference products N1~N6 of the enterprise were all negative.

The detection limit of the kit testing company is 5copies/uL.

Repeatability: Test repeatability reference product CV≤10.0% (n=10).

Kit specificity: Use the kit to detect other respiratory pathogens such as influenza A virus, influenza B virus, adenovirus, respiratory syncytial virus, Klebsiella pneumoniae, norovirus, rotavirus, Epstein-Barr virus, cytomegalovirus Viruses, Streptococcus pneumoniae, rhinovirus, measles virus, Group B streptococcus, Salmonella, Shigella, and normal human throat swab samples were all negative.

【Precautions】

1. Samples from patients with enterovirus infection are infectious. The collection, storage, transportation, and processing of samples should follow the country’s corresponding management practices and biosafety regulations. When handling samples, relevant protective measures must be taken to ensure that the experimental staff Safety.

2. The results of this kit will be affected by factors such as the source of the sample itself, sample collection process, sample quality, sample transportation conditions, sample pretreatment, etc., as well as the quality of RNA extraction, the working status of the fluorescent quantitative PCR instrument, the operating environment, and the current molecule. Limitations such as the limitations of biological technology may lead to false positive or false negative test results. The user must understand the potential errors and accuracy limitations that may exist in the detection process.

3. The kit should be transported and stored at low temperature. Before using each reagent in the kit, fully melt and shake it evenly, then centrifuge it briefly. Avoid repeated freezing and thawing under unnecessary conditions.

4. All reagents in this kit have been specially formulated for the above detection. Random replacement of any reagents in the kit may affect the use effect. The components of different batches of kits cannot be mixed with each other.

5. Please strictly partition the experiment:

The first area: reagent preparation area-prepare the reagents needed for amplification;

Second area: sample processing area-sample processing to be tested;

The third area: detection area-amplification detection.

The relevant laboratory management regulations shall be strictly implemented in accordance with the management regulations on gene amplification testing laboratories promulgated by the administrative department.

The items in each area are for exclusive use and must not be cross-used to avoid contamination; please clean the workbench after the experiment. The lysate of samples stored below −18°C or −70°C should be thawed at room temperature before sample addition and used after short centrifugation.

6. ​​During the experiment, pay attention to prevent the contamination of the reagents by exogenous nucleic acid, and pay attention to the positive control operation after adding the sample RNA. It is recommended to use a separate, dedicated pipette and tip when preparing reaction reagents and adding RNA templates.

7. The reaction tube containing the reaction solution should be capped or put into a sealed bag and then transferred to the sample processing area.

8. When the reaction solution is aliquoted, it should be avoided to produce bubbles. Before using the machine, check whether the reaction tubes are tightly capped to prevent the leakage of fluorescent substances from contaminating the instrument.

9. The sample should be completely dropped into the reaction solution when adding the sample, and no sample should stick to the tube wall. The tube cover should be tightly closed as soon as possible after sample addition.

10. Take out the reaction tube immediately after amplification, seal it in a special plastic bag, and discard it at the designated place.

11. The centrifuge tube and tip used in the experiment must be free of RNase and DNase. The centrifuge tubes and Tip heads used in the experiment must be treated harmlessly. The tip used in the experiment should be directly driven into the waste tank containing 84 disinfectant, and sterilized together with other waste products before discarding.

12. The workbench and various experimental supplies are regularly disinfected with 75% alcohol or ultraviolet light.

13. All chemicals are potentially dangerous. During operation, please wear appropriate laboratory overalls and protective measures such as disposable gloves. The used kits are clinical wastes and should be disposed of properly.

【references】

Tao Jianping, Yang Sida, Deng Li, et al. Diagnosis and treatment of severe hand, foot and mouth disease[J]. Chinese Journal of Practical Pediatrics, 2009, 24(6):423-426.

"Hand, Foot and Mouth Disease Diagnosis and Treatment Guidelines (2018 Edition)" Compilation Expert Committee. Hand, Foot and Mouth Disease Diagnosis and Treatment Guidelines (2018 Edition)[J]. Chinese Journal of Infectious Diseases, 2018, 36(5):257-263.

Qiu Baoqiang, Yan Yunying. Research progress in the etiology of hand, foot and mouth disease[J]. Guangxi Medicine, 2016, 38(5):698-700.

Xiao X, He Y, Yu Y, et al. Simultaneous detection of enterovirus 71 and coxsackievirus A16 by multiplex real-time PCR with an internal control[J]. Acta Microbiologica Sinica, 2009.

【Basic Information】

Registrant/manufacturer name: Jiangsu Hongwei Tesi Pharmaceutical Technology Co., Ltd.

Address: No.888 Zhujiang Road, Jiugang Street, Rudong County, Jiangsu Province (Rudong High-tech Zone Life and Health Industrial Park)

After-sales service unit: Jiangsu Hongwei Tesi Pharmaceutical Technology Co., Ltd.

Contact information:

Tel: 0513-80562880

Fax: 0513-80562881

Zip code: 226400

Website: http://www.hongweitest.com/

Production address: No.888 Zhujiang Road, Jiugang Street, Rudong County, Jiangsu Province (1st Floor, Building 10, Life and Health Industrial Park, Rudong High-tech Zone)

production date, production batch number and expiry date until see the product box

[Medical Device Production License Number]

[Medical Device Registration Certificate Number/Product Technical Requirement Number]

[Instruction approval date and revision date]

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