SN/T 5108-2019Method for detecting HRMA of rotavirus, astrovirus, norovirus and Zaru virus in border port (English PDF)

9.1 Sample Collection

Collection of fecal specimens and vomit specimens was performed in accordance with Sn/T 1720.

9.2 Laboratory inspection

9.2.1 Sample pretreatment

Take a stool sample or vomit with a size of about 100 mg of soybeans, or a 100 µL water sample, add 900 µL of physiological saline, shake vigorously and mix to make a 10% suspension; then centrifuge at 8000 r/min at 4 ° C for 5 min; To a sterilized clean centrifuge tube. Not immediately During the test, the samples were stored frozen in a -70 ° C ultra-low temperature refrigerator for future use.

9.2.2 Viral nucleic acid extraction

Take 9.2.1 100 µL of the pretreated sample, add 1 mL of Trizol solution, shake and mix and leave it at room temperature for 5 minutes, then add.200 µL of chloroform, cover the centrifuge tube, shake vigorously by hand and mix for 15 seconds, and leave it at room temperature for 5 minutes. 4 Centrifuge at 2,000 r/min for 10 min at °C, take the upper aqueous phase to another centrifuge tube, add 500 µL isopropanol to precipitate RNA, mix thoroughly and leave at room temperature for 10 min, then centrifuge at 12,000 r/min for 10 min, discard the supernatant, and add 1 mL Wash once with 70% ethanol, centrifuge at 2,000 r/min for 5 min, and discard the supernatant was left at room temperature for 5 min to 10 min, and 50 µL of DEPC water (or other nucleic acid solubilizing solution) was added to dissolve the nucleic acid precipitates. After aliquoting, the nucleic acid was stored at -70 ° C and stored for later use.

9.2.2.2 Kit extraction nucleic acid method

Follow the commercial kit instructions for viral genome extraction.

9.2.3 Quadruple high-resolution melting curve detection

9.2.3.1 Primers

The primer sequences are shown in Table 1.

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Table 1 Primer sequences

Rotavirus 5'-GGGGTCTCCCAAAAAGGGTGTAGGTAGATCACGTRCTGTTGCSCGA-3'5'-GGTGGTCCCCCAAAAAGGTGGTAGTCTCCTVGGTAGGTAGATGTGTGTRTGRT Forward Reverse Astrovirus 5'-GGGGGTCCCCAAAAGGGGTTCAGGTAGAGCACGTCAT-3'5'-GGGGTTCCCCAAAAAGGTGTCGTCTTGTGGCACCCCGTGTCTCTGA-3 ' Forward Reverse Norovirus 5'-GGGGTCCCCAACAGGATCACGATCACCATCA-3GA'TA-3'5'-GGGGTGTCCCCAAAAGTGGTCAGTCA-CATCACAT Forward Reverse Zara virus 5'-GGGGGTCCCCAAAAGGGTCTCCGTCCCCCCCTCACRRAWGWGCBTGTGTTT- 3'5'-GGGGTTCCCCAAAAAPGGTCAGGTCCGGCRCTCCACAAVSCTCBCCC3 Forward Reverse Note. In degenerate bases, R is A or G; S is C or G; Y is C or T; V is A or C or G; W is A or T; B is G or T or C.

9.2.3.2 Standard, positive control, negative control and blank control settings

Standards, positive controls, negative controls and blank controls were set during the test. Standards are cloned rotavirus, astrovirus, Norovirus and Zarrovirus some nucleotide sequences (must include the amplified region) in vitro transcription and synthesis of RNA; positive control is reserved the nucleic acids of positive samples of these four viruses or RNA synthesized by in vitro transcription; negative controls are nucleic acids extracted from normal human stool; blank the control was sterile water.

9.2.3.3 Reaction system composition

The reaction system composition is shown in Table 2.

Table 2 Reaction system composition

Component volume

2 × onestepRT-PCRBuffer 10µL Rt-pcrenmememix 0.4µL Rotavirus forward and reverse primers (20 µM) 0.25 µL each Astrovirus forward and reverse primers (20 µM) 0.1 µL each Norovirus forward and reverse primers (20 µM) 0.12 µL each Zarau virus forward and reverse primers (20 µM) 0.18 µL each 20 × EVAGreen 1µL RNA template 4µL DEPC water 3.3µL 20 µL total volume Note. If other commercial kits have the same effect, they can also be used.

9.2.3.4 Reaction conditions

The reaction conditions were. reverse transcription at 42 ° C for 5 min; pre-denaturation at 95 ° C for 10 sec; denaturation at 95 ° C for 10 sec; annealing at 60 ° C for 30 sec;

Cycle; set temperature gradually increased from 65 °C to 95 °C, the rate of rise is 0.1 °C/cycle. Due to the different performance of different PCR instruments, SN/T 5108-201919 ????? ????, ????,?

The reaction conditions can be appropriately adjusted according to the condition optimization.

9.2.3.5 High-resolution melting curve analysis

Analyze the generated melting curve with the software that comes with the instrument, and change the melting curve of the positive control/negative control/blank control/sample.

Compare with the melting curve of the standard. The curve of the positive sample is a typical single peak, while the curve of the negative sample is all straight, without Melting peaks.

9.2.4 Judgment and report of test results

Only when the melting curve of the positive control is consistent with the standards, and the negative control/blank control are all in a straight line, the sample can be processed.

The results are judged and reported, otherwise the experiment is deemed to have failed and the experiment needs to be re-run. The test results of this standard are judged and reported as follows.

a) Check that the melting curve of the sample is the same as that of the rotavirus standard, and report "Detection of rotavirus-specific gene (HRMA law)";

b) The melting curve of the test sample is consistent with that of the astrovirus standard, and the report "Detection of astrovirus-specific gene (HRMA law)";

c) The melting curve of the test sample is consistent with that of the Norovirus standard, and the report "Norovirus-specific gene (HRMA) detected law)";

d) The melting curve of the test sample is the same as that of the Zaravirus standard, and the report "Detection of the Zaravirus Specific Gene (HRMA) law)";

e) The melting curve of the test sample is the same as that of the blank control, that is, when all lines are in line, the report "No rotavirus, astrovirus, Norovirus, Zaravirus specific genes (HRMA method). " SN/T 5108-201919 ????? ????, ????,?