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HPV (human papilloma virus) typing detection primers as well as detection method and application thereof

A technology of HPV66 and primer pairs, which is applied in biochemical equipment and methods, microbiological measurement/inspection, DNA/RNA fragments, etc., can solve the problems of inability to effectively apply clinical detection, low accuracy rate, and high quality requirements for personnel. Achieve rapid and accurate molecular detection methods, reduce inspection costs, and avoid cross-contamination

Inactive Publication Date: 2016-10-26
沈阳中科赛尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Advantages of this method: simple operation, low price, suitable for preliminary screening; disadvantages: hollow cells are the main morphological changes of HPV infection, but due to other viral infections and human factors, vacuoles or hollows may appear in the cells In addition, cytological examination is easily affected by factors such as sampling, staining, and subjective judgment of cytopathologists. Therefore, the application of cytopathological detection of HPV has low sensitivity, poor specificity, high false negative rate and false positive rate, and cannot detect HPV. type
Advantages of this method: simple operation, low price, suitable for preliminary screening; disadvantages: low accuracy, high quality requirements for personnel, severe pain for patients, and unwillingness to cooperate with the inspection due to psychological resistance
Advantages of this method: clear principle and relatively simple operation; disadvantages: the objects of serological detection are antigens and antibodies. Since the human body has a certain hysteresis in the immune response to HPV, serological detection is not suitable for those with no immune response and HPV latent infection. Will produce missed detection
Although inexpensive, dsDNA-conjugated fluorochrome luminescence assays have a significant disadvantage that their specificity is highly susceptible to non-specific gene amplification and primer-dimer formation, which makes dsDNA-conjugated fluorochromes qPCR The method has not been able to be effectively applied to clinical detection
Probe qPCR can effectively solve the non-specific problem of double-stranded DNA binding fluorescent dye qPCR, but its main disadvantage is that the cost of probe design is too high to be widely used in the screening of diseases such as HPV infection.

Method used

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  • HPV (human papilloma virus) typing detection primers as well as detection method and application thereof
  • HPV (human papilloma virus) typing detection primers as well as detection method and application thereof
  • HPV (human papilloma virus) typing detection primers as well as detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Primer design.

[0076] The primer-blast software on NCBI designed specific primers for different high-risk or low-risk HPV types to ensure that the primer pairs had no homology with other HPV types and human genomes. Afterwards, the obtained multiple pairs of primers (average 10 pairs) were input into the oligo 6 software for analysis, comprehensively considering the primer self-matching, neck loop structure, amplification efficiency and mismatching situation, and respectively obtained the optimal primers for different high-risk or low-risk types. Type HPV primer pair, as follows

[0077] The sequence of the high-risk HPV16-specific primer is shown in SEQ ID NO: 1-2:

[0078] SEQ ID NO: 1 (HPV16F): 5'---CAGACGACTATCCAGCGACC---3';

[0079] SEQ ID NO:2(HPV16R):5'---GCAGTGAGGATTGGAGCACT---3';

[0080] The sequence of the high-risk HPV18-specific primer is shown in SEQ ID NO: 3-4:

[0081] SEQ ID NO: 3 (HPV18F): 5'---CTGCTACACGACCTGGACAC---3';

[0082] SEQ ...

Embodiment 2

[0137] Example 2: Construction of plasmid standards and formulation of standard curves.

[0138] In order to test whether the designed primers can be accurately applied to the qPCR typing detection of HPV, the amplification standard curves of various primers are obtained correspondingly.

[0139] To obtain the standard curve of HPV53 as an example:

[0140] 1. Construction of 53-type plasmid standard:

[0141] (1) Target fragment amplification, using 53-type target fragment amplification primers (upstream: 5-GGTATCCGTATGGAGTGTG-3; downstream: 5-CCACCTTGCTACATACATGC-3), using HPV53-infected clinical sample DNA as a template. KOD-Plus-Neo (TOYOBO) kit was used for amplification, and the reaction system and reaction conditions are shown in Tables 1 and 2.

[0142] Table 1 Plasmid Construction Target Fragment Amplification System

[0143]

[0144] Table 2 Plasmid construction target fragment amplification reaction conditions

[0145]

[0146] (2) Gel recovery and purific...

Embodiment 3

[0162] Example 3: Detection of primer specificity.

[0163] In order to prove the amplification specificity of various specific primers to the target type, all specific primers in the invention were amplified with the DNA of clinical samples infected by 21 common individual HPVs, in which various clinical samples were tested by a third party to verify the infection Types of. And all have undergone positive reference amplification to ensure DNA concentration.

[0164] Now take type 16 primer as an example

[0165] 1. DNA extraction from clinical samples infected with 21 separate HPVs:

[0166] (1) Tighten the cap of the preservation tube containing the sample, and vortex for 15 seconds to suspend as much exfoliated cells on the cervical brush as possible in the preservation solution.

[0167] (2) Pipette 1ml of preservation solution into a 1.5ml EP tube, centrifuge at 8000g for 1 minute, and discard the supernatant.

[0168] (3) Use the protocol of QIAGEN (Germany) DNA MINI...

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Abstract

The invention relates to the technical field of biological detection, in particular to HPV (human papilloma virus) typing detection primers as well as a detection method and an application thereof. QPCR primers for typing detection of HPV are shown as SEQ ID NO:1-38; the invention also discloses an application of the qPCR primers for typing detection of the HPV in preparing an HPV detection kit. The specificity and the sensitivity of the primer pairs and the kit provided by the invention in HPV typing detection are greatly higher than that of a common reverse dot blot method, and the primer pairs and the kits are relatively low in requirements on environment and operation and are broad in application. Meanwhile, according to the invention, a DNA double-stranded chimeric fluorescent dye qPCR detection method is applied to the HPV typing detection; the detection specificity is improved based upon double analysis through an amplification curve and a melting curve, so that the specificity can reach the detection level of a probe method, and meanwhile, cost can be greatly reduced; the primers have an important significance for the promotion of HPV general survey and for the prevention and treatment of cervical carcinoma; and the primers are quite high in popularization and application values.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an HPV typing detection primer and a detection method and application thereof. Background technique [0002] Human papillomavirus (Human Papilloma Virus, HPV) belongs to the papillomavirus genus, is a virus that specifically infects the skin or mucosal stratified epithelium. HPV is a double-stranded DNA virus with a genome size of about 7900 base pairs. The genome encodes three functional regions, namely the early transcription region (Early Region, E region), the late transcription region (Late Region, L region) and the upstream regulatory region (LCR). The E region consists of 7 genes in sequence, E6, E7, E1, E2, E3, E4 and E5, which have the functions of participating in the replication, transcription, translation, regulation and cell transformation of viral DNA. The L region mainly includes capsid protein L1 and minor capsid protein L2, whose function is to enc...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/708C12Q1/686C12Q2600/16C12Q2527/107C12Q2537/143C12Q2545/113
Inventor 刘一博谢静娴田柳黄妙玲于丹
Owner 沈阳中科赛尔生物科技有限公司
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