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Constant-temperature fluorescence detection primer group, kit and detection method for avoiding false-negative hand-foot-mouth virus CoxA16

A technology for fluorescence detection and detection of primers, applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., can solve the problems of hand-foot-mouth virus CoxA16 hazards, complex detection samples, etc., and achieve the probability of primer-dimer Low, easy to identify, high specificity effect

Inactive Publication Date: 2016-01-06
GUANGDONG NO 2 PROVINCIAL PEOPLES HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the test samples are complicated, and because the current detection method does not adopt monitoring methods for false negative test results, there is a possibility of missed detection, but hand, foot and mouth virus CoxA16 is seriously harmful, and the harm caused by missed detection is immeasurable. In addition, PCR amplification requires expensive Many basic testing units do not have the corresponding testing conditions

Method used

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  • Constant-temperature fluorescence detection primer group, kit and detection method for avoiding false-negative hand-foot-mouth virus CoxA16
  • Constant-temperature fluorescence detection primer group, kit and detection method for avoiding false-negative hand-foot-mouth virus CoxA16
  • Constant-temperature fluorescence detection primer group, kit and detection method for avoiding false-negative hand-foot-mouth virus CoxA16

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 The establishment of hand-foot-mouth virus CoxA16 constant temperature fluorescence detection kit

[0062] The constant temperature fluorescent detection kit of hand, foot and mouth virus CoxA16, including detection primer set, internal standard primer set, reaction solution, Bst DNA polymerase, AMV Reverse transcriptase, positive control, negative control and internal standard.

[0063] (1) Detection primer set: The CoxA16CAA gene of hand, foot and mouth virus is used as the target gene to design primers. The detection primer set includes a pair of outer primers, a pair of inner primers and a pair of loop primers. The nucleotide sequences are as follows: Show:

[0064] CAA-F3: 5'-AATGCTAGTGACAAGAATCTCA-3';

[0065] CAA-B3: 5'-GTTTGGCTACGACAAATGTG-3';

[0066] CAA-FIP: 5'-AAACCGGCACGGCTAAAGAAGTGTTGAACCATCACTCCA-3';

[0067] CAA-BIP: 5'-AGCATCATTACAATGCCCACCAATATCCCATCAAGTCAATGTCC-3';

[0068] CAA-LF: 5'-ATTCCCAATGGCTGTCTCC-3';

[0069] CAA-LB: 5'-...

Embodiment 2

[0081] The constant temperature fluorescent detection method of embodiment 2 hand-foot-mouth virus CoxA16

[0082] Utilize the constant temperature fluorescent detection kit of the hand-foot-mouth virus CoxA16 of embodiment 1 to detect the sample, the steps are as follows:

[0083] (1) Extraction of RNA from samples to be tested: Extract RNA from samples using a viral RNA extraction kit.

[0084] (2) Using the primer composition of claim 1 to carry out reverse transcription and constant temperature amplification of the sample RNA to be tested:

[0085] The 25μl reaction system for constant temperature amplification contains: CAA-F30.2μM, CAA-B30.2μM, CAA-FIP1.6μM, CAA-BIP1.6μM, CAA-LF0.8μM, CAA-LB0.8μM, CAB-F30.2μM , CAB-B30.2μM, CAB-FIP1.6μM, CAB-BIP1.6μM, CAB-LoopF0.8μM, CAB-LoopB0.8μM, reaction solution 12.5μl, Bst DNA polymerase 8U, AMV Retrovirus 0.8U, 10×SYBRGreenI0.5μl, sample to be tested 2μl, internal standard 2μl, make up to 25μl with ultrapure water;

[0086]...

Embodiment 3

[0092] Embodiment 3 Sensitivity experiment

[0093] Hand-foot-mouth CoxA16 virus RNA is carried out 10 times gradient dilution, carries out sensitivity experiment as template, detects with the method for embodiment 2, determines the minimum detection limit of internal standard gene by sensitivity experiment 10 2 copies / ml (eg figure 1 shown), and review the minimum detection limit ( figure 2 ), and set the internal standard concentration as the concentration of the lowest detection limit.

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Abstract

The invention discloses a constant-temperature fluorescence detection primer group, a kit and a detection method for avoiding false-negative hand-foot-mouth virus CoxA16. The primer group comprises a detection primer group and an internal standard primer group, wherein the detection primer group is designed according to a hand-foot-mouth virus CoxA16 CAA gene which has a sequence as shown in SEQ ID NO.1-6; and the internal standard primer group is designed according to a hand-foot-mouth virus CoxA16 CAB gene which as a sequence as shown in SEQ ID NO.7-12. Besides the primer groups, the constant-temperature detection kit for hand-foot-mouth virus CoxA16 further comprises DNA polymerase, a reaction liquid, reverse transcriptase, an internal standard target segment, positive control and negative control. The detection primer group, the kit and the detection method have the advantages of quickness, high efficiency, convenience in operation, high specificity, high sensitivity, field detection availability and the like, can be used for effectively avoiding occurrence of false negative, and is suitable for popularization and application.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to a primer set, a kit and a detection method for constant temperature detection of hand-foot-mouth virus CoxA16. Background technique [0002] Hand, foot and mouth disease is a global infectious disease, and there have been reports of the epidemic in most countries and regions over the years. It is mainly a common infectious disease caused by enteroviruses, and is mainly transmitted through the fecal-oral route and close contact. According to epidemiological surveys, children under the age of 5 are the high-risk group of the disease, accounting for more than 90% of the total number of cases. Among them, infants under the age of 3 are the high-risk group of severe disease, and the probability of adult infection is extremely low. Hand, foot and mouth disease is widely distributed in epidemic areas, and there is no strict regionality. Outbreaks have oc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6844
Inventor 曹东林胡亮杉林茂锐石磊周旋钟丽梅方晓琳陈洵唐大运张璜邓梓欣雷达张知洪田军章
Owner GUANGDONG NO 2 PROVINCIAL PEOPLES HOSPITAL
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