CN104651531B - Kit for detecting juvenile recurrent respiratory papilloma virus - Google Patents

Abstract

The invention discloses a highly specific primer pair for detecting the juvenile recurrent respiratory papilloma HPV11 subtype, and a kit for one-step TapMan fluorescence quantitative PCR detection of the juvenile recurrent respiratory papilloma HPV11 subtype. The sequences of the highly specific primer pair for detecting the juvenile recurrent respiratory papilloma HPV11 subtype are shown in SEQ ID NO.1 and SEQ ID NO.2. A probe with the sequence of SEQ ID No.3 is contained in the kit. The kit is capable of quantitatively amplifying and detecting the juvenile recurrent respiratory papilloma HPV11 subtype, reducing the pollution, and increasing the detecting accuracy, and is simple and rapid in operation and is suitable for the clinical rapid typing test and the virus epidemiological investigation.

Description

Kit for the detection of recurrent respiratory papillomavirus in adolescents

technical field

The invention relates to a virus TapMan fluorescent quantitative detection kit, specifically a one-step TapMan fluorescent quantitative PCR detection kit for detecting HPV11 subtypes of adolescent recurrent respiratory papilloma.

Background technique

Juvenile-onset recurrent respiratory papilloma (JO-RRP) specifically refers to papillomatosis that occurs in the upper and lower respiratory tracts, and is a highly recurrent disease caused by the human papillomavirus (HPV) virus. And invasive disease, can occur in any part of the respiratory tract, which is most likely to accumulate at the junction of squamous epithelium and ciliated columnar epithelium. The first symptom of JORRP patients is mainly hoarseness. The age of onset is as young as two weeks and as old as 14 years old. The course of the disease is diverse. Some children can relieve spontaneously, but most of them show infiltrating growth and require multiple operations or even larynx and tracheal stenosis Obstruction asphyxiation death, clinical treatment is very difficult.

HPV belongs to the Papillomavacuovirus A genus of the family Papovaviridae. It is a circular double-stranded DNA virus composed of 8000 bases, and there are about 200 subtypes. According to the genome structure, biological characteristics and pathogenicity of the virus, it can be divided into five categories, namely α, β, γ, μ and ν. The low-risk HPV6 and HPV11 belong to the α papillomavirus. The low-risk type is closely related to adolescent recurrent respiratory papillomatosis. Scholars at home and abroad have carried out related treatment research. However, no effective cell culture model or simple virus reproduction model has been established. It is difficult to carry out traditional inactivated vaccines or monitoring the development of vaccines.

Studies have shown that HPV subtypes are related to disease severity and clinical course, and there are differences in virulence between subtypes. Therefore, it is particularly important to quickly distinguish the HPV11 subtypes that cause recurrent respiratory papillomaviruses in adolescents clinically. The detection sensitivity of conventional PCR method is not high, and it is prone to false positives. The existing SYBRGreen fluorescence method for detecting venereal disease HPV11 subtype is a non-saturated dye, which cannot be fully saturated and embedded in the DNA double strand, which will cause inaccurate detection results.

Contents of the invention

The present invention provides a pair of highly specific primers and probes that can be used to detect HPV11 subtypes of recurrent respiratory papilloma in adolescents, as well as a specific, sensitive, rapid and convenient method that includes the highly specific primers and probes. A kit for the quantitative detection of HPV11 subtypes in adolescent recurrent respiratory papilloma.

The sequences of the highly specific primer pair for detecting juvenile recurrent respiratory papillomavirus of the present invention are SEQ ID No.1 and SEQ ID No.2 respectively.

The highly specific sequence included in the kit for detecting juvenile recurrent respiratory papillomavirus of the present invention is the probe of SEQ ID No.3. For the convenience of use, there are also all experimental reagents needed for amplification such as 2×One Step RT-PCRBuffer III, TaKaRa Ex Taq HS (5U/μL) and PrimeScript RT Enzyme Mix II in the kit of the present invention, and only need to follow the instructions for detection Just add the test sample.

The kit for detecting juvenile recurrent respiratory papilloma virus of the present invention also contains as a negative control a DNA sample that does not contain the subtype of juvenile recurrent respiratory papilloma HPV11 and as a positive control a DNA sample containing juvenile recurrent respiratory papilloma DNA samples of HPV11 subtype.

The preferred amplification conditions for the aforementioned primer pair of the present invention are: 95° C. for 10 sec, 1 cycle → 95° C. for 10 sec, 55° C. for 30 sec, 72° C. for 30 sec, 40 cycles.

The kit of the present invention is actually a kit for one-step TapMan fluorescent quantitative PCR detection. One-step TapMan fluorescent quantitative PCR detection is to use the TapMan probe with a fluorescent substance at the 5' end and a quenching substance at the 3' end for fluorescence detection. When the probe is intact, the fluorescent substance at the 5' end is quenched by the 3' end Material constraints, can not emit fluorescence. When the TapMan probe is decomposed, the fluorescent substance at the 5' end will dissociate and emit fluorescence. In the PCR reaction, the specific probe and the template hybridize, and the extension is that the polymerase activity can decompose the fluorescent probe hybridized with the template, and the free fluorescent substance emits fluorescence. By detecting the fluorescence intensity in the reaction system, the purpose of detecting the amplification amount of the PCR product can be achieved. Real Time PCR reaction using this kit in PCR can be carried out continuously in the same reaction tube, which is easy to operate and can effectively prevent pollution.

Utilizing the kit of the present invention to detect the HPV11 subtype of adolescent recurrent respiratory papilloma by one-step TapMan fluorescent quantitative PCR detection has the advantage that the L1 gene conservation of the popular strain of HPV11 subtype of adolescent recurrent respiratory papilloma involves two highly specific genes. Primers and probes, on the basis of the DNA extraction of the virus, perform fluorescence quantitative PCR amplification on the tissue samples, and realize the quantitative detection of HPV11 subtypes of adolescent recurrent respiratory papilloma.

The TaqMan probe method used in the present invention is a probe designed for the specific nucleotide sequence of the adolescent recurrent respiratory papillomavirus HPV11 subtype. The probe is highly specific to the template, and the specificity and sensitivity are high. . It fills the blank of clinical rapid typing detection of recurrent respiratory papillomavirus HPV11 subtype in adolescents.

In the kit of the present invention, highly specific primers and probes are optimally designed according to conserved regions after alignment of the HPV11 subtype L1 gene sequence of adolescent recurrent respiratory papilloma. When the kit of the present invention is used for detection, quantitative PCR amplification is directly carried out after extracting the DNA of adolescent recurrent respiratory papilloma HPV11 subtype, and the present invention optimizes the reaction system and reaction conditions to make detection PCR amplification It is completed in one step, which simplifies the operation steps, reduces the chance of contamination, and improves the accuracy of detection. The one-step TapMan fluorescence quantification can directly perform the standard curve quantification of the initial virus copy number after the amplification is completed. At the same time, its operation is simple and fast, very It is suitable for the application of typing detection and epidemiological investigation of the virus.

Description of drawings

Figure 1: The electrophoresis diagram of the enzyme digestion identification of the recombinant plasmid containing the HPV11 subtype L1 gene sequence of adolescent recurrent respiratory papilloma,

Among them: M: 2000bp DNA Marker

1: Identification of recombinant plasmid pMD-HPV11-L1 by double enzyme digestion

2: Linearization of the recombinant plasmid pMD-HPV11-L1 by single enzyme digestion

Figure 2: The amplification curve of HPV11 one-step TapMan fluorescent quantitative PCR, where: the ordinate represents the amount of fluorescence, and the abscissa represents the number of cycles. The amplification curve from left to right indicates the copy number of the target DNA is 2.59×10 ⁸ -2.59× 10 ³ ;

Figure 3: One-step TapMan fluorescent quantitative PCR detection standard curve for HPV11 virus, where: the vertical axis represents the Ct value. The abscissa represents the copy number of the sample, the correlation coefficient R2: 0.999, the amplification efficiency Eff: 101.1%;

Figure 4: is the amplification curve specific to the detection method;

Figure 5: The amplification curve of the sensitivity of the detection method, where: the ordinate represents the amount of fluorescence, the abscissa represents the number of cycles, and the copy numbers of DNA from left to right in the amplification curve are 1×10 ⁸ -1×10 ¹ .

detailed description

Below in conjunction with specific example and accompanying drawing description, the present invention will be further described

1. Design and synthesis of fluorescent quantitative PCR primers for adolescent recurrent respiratory papilloma HPV11 subtype

After alignment of the known HPV11 subtype L1 gene sequence of juvenile recurrent respiratory papilloma, primers and probes were designed using Primer Express 3.0 software according to the conserved region, and synthesized by Songbao Bioengineering (Dalian) Co., Ltd. The upstream and downstream primers and probes are:

SEQ ID No.1: (HPV11-F) 5'-TCCTCCTAAACCCTGTATC-3'

SEQ ID No.2: (HPV11-R) 5'-TGCTGGCATGATAAAATATG-3'

SEQ ID No.3: (HPV11-Probe) 5'-CATAAGCATCCGTGGCAACAACTT-3' (5'FAM, 3'TAMRA)

2. Preparation of Standards

Referring to the sequence of HPV11 subtype of juvenile recurrent respiratory papilloma registered on NCBI, after comparative analysis, the upstream and downstream primers that can amplify the partial sequence of L1 gene were designed and synthesized by Songbao Bioengineering (Dalian) Co., Ltd. The upstream and downstream primers are:

SEQ ID No.4:

(HPV11-5674-F) 5'-TGGACGACCAGCTCTTCCTTAAACAC-3';

SEQ ID No.5:

(HPV11-6807-R)

5'-AGTGCAAGTCTGGGTTGCAGCCAACA-3'.

Take the respiratory papilloma tissue in a biological safety cabinet, grind and dilute, and extract the DNA of the tissue according to the instructions of the Omega DNA Extraction Kit (Cat. No.: D3096) (the used utensils and consumables must be strictly sterilized), and then use the one-step PCR method of TaKaRa Company The operation of the kit is as follows:

Use the DNA extract as a template for PCR amplification to prepare a 25 μl system,

2×1 step buffer (including dNTP) 13μL

PrimeScript 1 Step Enzyme Mix 1μL

Upstream primer (HPV11-5674-F) 0.5 μL

Downstream primer (HPV11-6807-R) 0.5 μL

DNA extract (template) 1 μL

DNAse Free dH2O 9μL

Gently mix, centrifuge at low speed and place in a PCR automatic amplification instrument. Amplify according to the following program: 50°C for 30 minutes, 94°C for 2 minutes → 94°C for 30 sec, 52°C for 30 sec, 72°C for 45 sec for 35 cycles. The PCR product was purified and recovered by 1% agarose gel electrophoresis, ligated with pMD-20-T Vector overnight at 16°C with T4 ligase, transformed into DH5α competent cells, and single-clonal spots were picked and expanded in LB (Amp+). The plasmid pMD-HPV11-L1 was purified by the extraction kit, identified by SpeI/EcoRI digestion, and the positive plasmid was confirmed, as shown in Figure 1. After identification by sequencing, a large amount of the successfully constructed positive plasmid pMD-HPV11-L1 was extracted and linearized with EcoRI single enzyme digestion, purified and determined for concentration, and then frozen at -80°C. Use the formula: (copies/μL)=[(ng number×10 ^-9 )×(6.02×10 ²³ )]÷(base number×340) to convert the copy number of the target DNA.

3. Establishment of standard curve

According to the copy number obtained after quantification, the standard sample was made into six serially diluted detection standard products. In order to reduce the error and improve the repeatability of the experiment, each dilution sample was provided with three replicate holes to determine the Ct value ( Ct is cyclethreshold, which refers to the number of cycles experienced when the fluorescent signal in the reaction tube reaches the set threshold), so as to determine the standard curve with the best amplification effect.

(1) Fluorescent quantitative PCR amplification

The amplification system is 25 μL, which contains 2×One Step RT-PCR Buffer Ⅲ (including dNTP Mixture, Mg2+), TaKaRa Ex Taq HS (5U/μL), PrimeScript RT Enzyme Mix Ⅱ, DNAse Free dH2O, upstream primer SEQ1, downstream primer SEQ2 , probe SEQ3, and 2 μL of diluted standard.

Agilent Technologies-Mx3005p PCR amplifier was used for PCR amplification, and the program was: 95°C 10sec, 1 cycle (reverse transcription), 95°C 10sec, 55°C 30sec, 72°C 30sec, 40 cycles (PCR amplification)

The fluorescent signal collection was set at the end of 30 sec at 72°C in each cycle.

(2) Preparation of standard curve

The analysis of the PCR fluorescence signal was carried out by Agilent MxPro software, and the obtained fluorescence quantitative PCR amplification curve and the corresponding standard curve are shown in Figures 2 and 3, respectively.

4. Specific detection

The following common disease pathogens: mucosal low-risk HPV6, mucosal high-risk HPV16, skin low-risk HPV1, and skin high-risk HPV5 were used to detect the specificity of the established method. The results obtained by fluorescent quantitative PCR are shown in Figure 4. The results show that this method can only specifically amplify the positive control of HPV11 subtype DNA of adolescent recurrent respiratory papilloma, and the specificity is good.

5. Sensitivity detection

The standard sample was made into 8 dilutions of 1×10 ⁸ -1×10 ¹ (copies/μL), and the sensitivity of the established method was tested. The obtained fluorescent quantitative PCR results are shown in Figure 5. In order to ensure the accuracy of the detection, high-pressure sterilized water is set as the background value control, and the samples with ≤34 cycles and the curve with obvious exponential growth period are judged as positive. This fluorescence quantitative detection method can accurately detect about 10 copies of pathogens, while ordinary PCR intelligently detects 1×10 ⁴ copies, so it can be seen that the detection sensitivity of the fluorescent quantitative PCR of the present invention is 10 ³ times that of ordinary PCR, and the sensitivity is good.

<110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences

<120> Kit for detection of recurrent respiratory papillomavirus in adolescents

<160> 5

<210> 1

<211> 18

<212>DNA

<213> Artificial sequence (forward primer)

<400>

tcctcctaac cctgtatc 18

<210> 2

<211> 20

<212>DNA

<213> Artificial sequence (reverse primer)

<400>

tgctggcatg ataaaatatg 20

<210> 3

<211> 20

<212>DNA

<213> Artificial sequence (probe primer)

<400>

cataagcatc cgtggcaaca actt 24

<210> 4

<211> 26

<212>DNA

<213> Artificial sequence (upstream primer HPV11-5674-F to amplify partial sequence of L1 gene)

<400>

tggacgacca gctcttcctt aaacac 26

<210> 5

<211> 26

<212>DNA

<213> Artificial sequence (downstream primer HPV11-6807-R for amplifying partial sequence of L1 gene)

<400> agtgcaagtc tgggttgcag ccaaca 26

Claims (5)

1. A highly specific primer pair for detecting juvenile recurrent respiratory papillomavirus, characterized in that the primer pair sequences are SEQ ID No.1 and SEQ ID No.2 respectively. 2. The test kit for detecting juvenile recurrent respiratory papillomavirus is characterized in that the primers and sequences of SEQ ID No.1, SEQ ID No.2 and the probe of SEQ ID No.3 are included in the test kit . 3. the test kit for detecting juvenile recurrent respiratory papillomavirus according to claim 2, characterized in that there are also 2 × One Step RT-PCR Buffer III, TaKaRa Ex Taq HS (5U/μL) in the test kit And all experimental reagents needed for the amplification of PrimeScript RT Enzyme MixⅡ. 4. according to claim 2 or 3 described test kits for detecting the juvenile recurrent respiratory papilloma virus, it is characterized in that there is also a non-juvenile recurrent respiratory papilloma HPV11 subtype as negative control in the test kit DNA samples and a DNA sample containing the HPV11 subtype of juvenile recurrent respiratory papilloma as a positive control. 5. The pair of highly specific primers according to claim 1, wherein the amplification conditions are: 95° C. for 10 sec, 1 cycle → 95° C. for 10 sec, 55° C. for 30 sec, 72° C. for 30 sec, 40 cycles.