PCR (polymerase chain reaction) primer, kit and method for selectively amplifying RNA (ribose nucleic acid) from total nucleic acid of HBVs (hepatitis B viruses)
A hepatitis B virus, selective technology, applied in the field of PCR primers for selective amplification of RNA, can solve problems such as high cost and long time consumption
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Embodiment 1
[0026] Embodiment 1: HBV viral RNA standard substance detection
[0027] HBV RNA standard preparation. HBV plasmid DNA with T7 promoter is linearized and then transcribed in vitro to obtain single-stranded RNA. By calculating the copy number, then, dilute the RNA to 2e5 copies / μl, 2e4 copies / μl, 2e3 copies / μl, 2e2 copies / μl, 20 copies / μl, 2 copies / μl, 0.5 copies / μl, 0.125 copies / μl .
[0028] For quantitative PCR detection, a kit (One step PrimeScript RT-PCR Kit product number: RR064A) was used. The reaction system is shown in Table 1.
[0029] Table 1 reaction system
[0030]
volume
2×one-step RT-PCR buffer
10μl
Upstream primer (10μM)
0.6μl
Downstream primer (10μM)
0.6μl
Taqman probe (10uM)
0.2μl
PrimeScript RT Enzemy Mix II
0.4μl
Takara Ex Taq (5U / μl)
0.4μl
ROX(50×)
0.4μl
h 2 o
2.4μl
template
5μl
total capacity
20μl
[0031] The quantitative ...
Embodiment 2
[0049] Example 2: Detection of specific RNA in serum of HBV infected patients.
[0050] Extraction of total HBV nucleic acid: select serum (6 samples) of HBV patients whose DNA concentration is known. 200ul of HBV patient serum was extracted with Qiagen Viral RNA Extraction Kit (QIAamp Viral RNA Mini Kit product number: 52904), the elution volume was 100μl, and 10μl of the eluted nucleic acid was retained.
[0051] Quantitative PCR detection: The detection is divided into two types: HBV DNA and HBV RNA. Using the same detection kit (Onestep PrimeScript RT-PCR Kit product number: RR064A), the reaction system is shown in Table 2. The only difference is that PrimeScript RT Enzemy Mix II is not added to the wells for detecting HBV DNA alone, that is to say, no reverse transcription is performed. Add PrimeScript RT Enzemy Mix II only for detection of HBV RNA. At the same time, in order to detect the residual DNA in the HBV RNA sample.
[0052] Table 2 reaction system
[0053] ...
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