CN103255235B

CN103255235B - Real-time PCR (polymerase chain reaction) non-diagnostic detection method of panda source rotavirus CH-1 strain

Info

Publication number: CN103255235B
Application number: CN201310201711.0A
Authority: CN
Inventors: 颜其贵; 王成东; 马磊; 郭玲; 曹三杰; 文心田; 张志和; 王彬; 黄小波
Original assignee: Sichuan Agricultural University
Current assignee: Chengdu Research Base of Giant Panda Breeding; Sichuan Agricultural University
Priority date: 2013-05-27
Filing date: 2013-05-27
Publication date: 2015-04-22
Anticipated expiration: 2033-05-27
Also published as: CN103255235A

Abstract

The invention discloses a real-time PCR (polymerase chain reaction) non-diagnostic detection method of a panda source rotavirus CH-1 strain. An upstream primer F: 5'-AGTTATACGCAGCACGGACC-3' is adopted, and a downstream primer R: 5'-GTTGTTGCGTTTGGGTGTGGT-3 is adopted; and primer concentration is 0.5mumol/L, and circulation conditions of a real-time PCR process comprise that predegeneration is carried out for 30s at the temperature of 95 DEG C, degeneration is carried out for 5s at the temperature of 94 DEG C, annealing is carried out for 30 seconds at the temperature of 60.5 DEG C, 40 cycles are carried out, and a fluorescent signal after each cycle is detected. The minimum copy number detected by the fluorescent quantitative RT-PCR method established by the invention is 1.0*100 copies/micro litre and is at least 100 times higher than sensitivity of the conventional RT-PCR.

Description

The Real-time PCR nondiagnostic detection method of giant panda source rotavirus CH-1 strain

Technical field

The invention belongs to microbial technology field, relate to the Real-time PCR nondiagnostic diagnostic method for the rotavirus CH-1 strain of giant panda source.

Background technology

Rotavirus (Rotavirus, RV) is one of important pathogen causing infant and multiple young animal diarrhoea, is also important Zoonosis disease pathogen, worldwide loses huge because humans and animals infects RV every year.In the world, diarrhoea is the second largest factor causing Infant and child deaths, and RV accounts for space of top prominence in this factor.According to Zou Xing Huaihe River and Sun Feilong, it is that disease is lethal that giant panda is tending towards one of the main reasons in imminent danger, and in the various diseases of lethal giant panda, the most serious with intestinal tract disease.Ye Zhiyong finds that the sickness rate of digestive system is only second to parasitosis when adding up 50 routine field morbidity giant pandas, accounts for 20%, occupies Giant Panda sickness rate second place.1993, find that the giant panda of wherein 37% all dies from intestinal tract disease when Qiu Xianmeng etc. carry out systematic review to 102 dead giant pandas, as can be seen here, the harm of intestinal tract disease to Giant Panda Population can not be ignored.

The reported first giant pandas such as Wang Chengdongs in 2008 are because infecting rotavirus (Giant Panda Rotavirus, GPRV) children's giant panda (5 ~ 11 monthly age) intractable diarrhoea in age is caused, finally cause multiple organ failure and dead, and be successfully separated and obtain a strain giant panda source rotavirus CH-1.Because the height latent of GPRV brings great harm to the existence of this world's endangered rare animal of giant panda and breeding.As animal for display, giant panda and people's touch opportunity more, therefore GPRV is probably transmitted to people by giant panda, and makes infant ill, therefore, significant in Disease epizootic to the detection of giant panda rotavirus.

At present, the detection method of RV mainly contains virus culture, ELISA, electron microscopic observation, PAGE electrophoresis and RT-PCR.Due to the genotype non-cell pathogenicity of some RV, thus make first to be separated to obtain virus, then be affected by the recall rate of this method of immunological method identifying virus; The result of ELISA affects comparatively large by manual operation, easily occur false positive; PAGE method then not easily ensures the integrity of RNA; Electron microscopy test operation is loaded down with trivial details and susceptibility is not high; Conventional RT-PCR easily pollutes, need electrophoresis easily to make mistakes after amplification and can not carry out quantitatively, and fluorescent quantitation method then effectively can make up their deficiency with this several method compared with, and test-results judges special, responsive and quick.

Summary of the invention

Technical problem to be solved by this invention is the Real-time PCR nondiagnostic detection method providing the rotavirus CH-1 strain of a kind of giant panda source for the deficiencies in the prior art.

Technical scheme of the present invention is as follows:

A Real-time PCR nondiagnostic detection method for giant panda source rotavirus CH-1 strain, upstream primer F:5 '-AGTTATACGCAGCACGGACC-3 ', downstream primer R:5 '-GTTGTTGCGTTTGGTGTGGT-3; Primer concentration is 0.5 μm of ol/L, Real-time PCR cycling condition: 95 DEG C of denaturation 30s, 94 DEG C of sex change 5s, and 60.5 DEG C of annealing 30s, carry out 40 circulations, detect fluorescent signal after each circulation.

The minimum copy number that the fluorescent quantitative RT-PCR method that the present invention sets up detects is 1.0 × 10 ⁰copy/μ L (see Fig. 5), at least exceeds 100 times than the sensitivity of conventional RT-PCR (see Fig. 6 and table 1).

Accompanying drawing explanation

Fig. 1 GPRV CH-1 object segment pcr amplification, M:DL2000DNA relative molecular mass standard; 1:PCR amplified production;

The fixed detected result to different concns plasmid of Fig. 2 fluorescence (along transverse axis parallel lines from left to right curve sequence number be followed successively by 1,2,3,4,5);

Fig. 3 typical curve;

Fig. 4 specific amplification curve, 1: giant panda rotavirus CH-1 strain; 2: epidemic diarrhea virus; 3: Transmissible gastroenteritis virus; 4: intestinal bacteria; 5: Salmonellas;

Fig. 5 fluorescence quantitative RT-RCR detected result, the plasmid concentration that curve 1 ~ 8 is corresponding is respectively 1.0 × 10 ⁷, 1.0 × 10 ⁶, 1.0 × 10 ⁵, 1.0 × 10 ⁴, 1.0 × 10 ³, 1.0 × 10 ², 1.0 × 10 ¹, 1.0 × 10 ⁰copy/μ L;

Fig. 6 conventional RT-PCR detected result ,-: negative control; +: positive control; M:DL2000DNA relative molecular mass standard; 1 ~ 8: corresponding plasmid concentration is 1.0 × 10 respectively ⁷, 1.0 × 10 ⁶, 1.0 × 10 ⁵, 1.0 × 10 ⁴, 1.0 × 10 ³, 1.0 × 10 ², 1.0 × 10 ¹, 1.0 × 10 ⁰copy/μ L;

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.

1.1 giant panda rotavirus vp 4 gene order amplifications

According to GPRV VP4 sequence (HQ641296) in GenBank, Oligo6.0 and Primer5.0 is used to design a pair fluorescence quantification PCR primer: upstream primer F:5 '-AGTTATACGCAGCACGGACC-3 ', downstream primer R:5 '-GTTGTTGCGTTTGGTGTGGT-3, between amplification rotavirus vp 4 gene conserved regions, size is 107bp, and primer send and synthesized by Shanghai Invitrogen company.

The preparation of 1.2 standard positive templates

1) extracting of viral RNA and the extraction of DNA of bacteria

Get 200 μ L giant panda RV CH-1 strain virus liquid, according to the operation of OMEGA company RNA Isolation Kit specification sheets, extracting viral RNA ,-70 DEG C of preservations.

2) reverse transcription

Using the giant panda source rotavirus CH-1 strain RNA of extracting as template, illustrate by PrimeSccript RT reagent Kit, adopt 10 μ L systems as table 1:

Table 1

Carry out reverse transcription reaction by following program: 37 DEG C of 15min, 85 DEG C of 5s, 4 DEG C of preservations, obtain viral cDNA.

3) amplification of goal gene

PCR reaction system is 50 μ L, and composition is as table 2:

Table 2

Increase according to following program: 95 DEG C of denaturation 5min, 95 DEG C of circulation sex change 30s, 60.5 DEG C of annealing renaturation 30s, 72 DEG C extend 30s, and after carrying out 30 circulations, 72 DEG C of ends extend 10min, 4 DEG C of preservations.Get amplified production electrophoresis in 10g/L sepharose, observations (Fig. 1).

4) cloning and identification of PCR primer

After agarose gel electrophoresis, cut object fragment Gel Extraction Kit purifying and reclaim, after being connected with pMD-19TSimple Vector, transform DH _{5 α}competent cell.The single bacterium colony of picking after the dull and stereotyped upper 37 DEG C of cultivation 12 ~ 16h of the LB containing penbritin (Amp), plasmid is extracted with Endo-free Plasmid Mini Kit after increasing bacterium, carry out pcr analysis qualification, by screening positive recombinant plasmid serve Hai Ying fine horse Bioisystech Co., Ltd order-checking qualification, the positive plasmid including correct goal gene is stored in-70 DEG C for subsequent use.

The optimization of 1.3 quantitative fluorescent PCR reaction conditionss

Take positive plasmid as standard substance, respectively to primer concentration (0.4 ~ 1.0 μm of ol/L), annealing temperature (55 ~ 65 DEG C), cycle number (35 ~ 45 circulations) is optimized, and judges optimum result by comparing Ct value, melt curve analysis, fluorescence intensity and amplification efficiency.Through test conditions screening, obtaining best primer concentration is 0.5 μm of ol/L, and optimum cycle condition is: 95 DEG C of denaturation 30s, 94 DEG C of sex change 5s, and 60.5 DEG C of annealing 30s, carry out 40 circulations, detect fluorescent signal after each circulation.

The drafting of 1.4 typical curves

Extract positive recombinant plasmid, in nucleic acid-protein analyser, measure OD260 and OD280 respectively, according to plasmid copy number calculation formula, the DNA copy number calculated in plasmid is about 6.4 × 1013 copy/μ L.10 times of doubling dilutions are carried out successively to positive recombinant plasmid, gets 5 dilution recombinant plasmids as standard substance template, utilize the quantitative fluorescent PCR reaction conditions optimized, drawing standard curve.

1) kinetic curve

As shown in Figure 2, the cycle threshold corresponding to curve 1,2,3,4,5 is 8.96,11.93,15.56,18.27,21.50 respectively.

The plasmid concentration that curve 1,2,3,4,5 is corresponding is respectively 6.4 × 10 ¹³, 6.4 × 10 ¹², 6.4 × 10 ¹¹, 6.4 × 10 ¹⁰, 6.4 × 10 ⁹copy/μ L;

2) standard form

As shown in Figure 3, the straight-line equation of typical curve is: y=-3.142x+52.254, and wherein y is cycle threshold, and x is plasmid template concentration.

1.5 specific detection

Select common animals dysentery cause of disease epidemic diarrhea (PEDV), transmissible gastroenteritis (TGEV), intestinal bacteria, Salmonellas, detect by the fluorescent quantitation method set up, the specificity of checking the method.The fluorescent quantitation method amplification that test is set up shows to only have giant panda rotavirus CH-1 strain to have fluorescent signal, and other virus and the equal unstressed configuration signals (as Fig. 4) of bacterium, illustrate that the method specificity is better.

1.6 repeatability detect

Detect test repeatability and the stability of GPRV to assess the method, select the standard plasmid of high, medium and low three different concns to be that template is tested, each sample repeats 3 times.Replica test result shows, the error repeatedly increased is less than 1 circulation, illustrates that the repeatability of the method is high, ensure that the reliability of different sample detection result.

1.7 compare with conventional RT-PCR susceptibility

Adjusting positive recombinant plasmid concentration, is 1.0 × 10 through 10 times of doubling dilutions to concentration ⁰copy/μ L, gets 1.0 × 10 ⁷, 1.0 × 10 ⁶, 1.0 × 10 ⁵, 1.0 × 10 ⁴, 1.0 × 10 ³, 1.0 × 10 ², 1.0 × 10 ¹, 1.0 × 10 ⁰the plasmid of a copy/μ L8 concentration is template, and the reaction conditions obtained with this test carries out fluorescence quantitative RT-RCR amplification, and compared with conventional RT-PCR method.

The minimum copy number that the fluorescence quantifying PCR method that the present invention sets up detects is 1.0 × 10 ⁰copy/μ L (see Fig. 5), at least exceed 100 times than the sensitivity of conventional RT-PCR (see Fig. 6 and table 1), the comparing result of two kinds of methods is in table 1.

Table 3 fluorescence quantitative RT-RCR and conventional RT-PGR method remolding sensitivity are comparatively

Plasmid template copy number	Real-time PCR cycle threshold	Conventional RT-PCR
			1.0×10 ⁷	20.23	+
1.0×10 ⁶	20.77	+
			1.0×10 ⁵	21.56	+
1.0×10 ⁴	22.36	+
			1.0×10 ³	25.83	+
1.0×10 ²	26.35	+
			1.0×10 ¹	28.07	-
1.0×10 ⁰	29.90	-

+: represent positive;-: represent negative.

Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.

Claims

1. a Real-time PCR nondiagnostic detection method for giant panda source rotavirus CH-1 strain, is characterized in that, upstream primer F:5 '-AGTTATACGCAGCACGGACC-3 ', downstream primer R:5 '-GTTGTTGCGTTTGGTGTGGT-3; Primer concentration is 0.5 μm of ol/L, Real-time PCR cycling condition: 95 DEG C of denaturation 30s, 94 DEG C of sex change 5s, and 60.5 DEG C of annealing 30s, carry out 40 circulations, detect fluorescent signal after each circulation.