Kit, probe and primer combination for rapidly detecting multiple respiratory pathogens
A respiratory and pathogen technology, applied in the field of kits, probes and primer combinations for rapid detection of various respiratory pathogens, which can solve the problems of reduced throughput, long operation time, and cumbersome operation, avoiding false negatives and false positives. , Improve the effect of detection throughput
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Embodiment 1
[0028] Example 1 Design of specific primers and probes
Download the ORF1ab and N genes of 2019-nCoV through NCBI search; the M gene of FluA and MPV; the N gene of FluB; the P gene of RSVA and RSVB; the 5'UTR gene of HRV; the HA gene of H3N2; the HEXON gene of HAdV3 and HAdV7 ; MP CARDS gene and RNaseP gene, use Beacon Designer or Primer 3 software to design specific amplification primers and molecular beacons or Taqman probes for the above pathogens, which are synthesized by Shanghai Shuoying Biotechnology Co., Ltd. The primers and probes used for detection are shown in Table 1:
Table 1 Primer and probe sequence information
Embodiment 2
[0029] Example 2 Sample nucleic acid extraction
(1) Collection, storage and transportation of clinical samples: applicable to throat swabs and sputum samples. Throat swab: Pass the swab across the base of the tongue, wipe the pharyngeal tonsils on both sides of the subject with slight force back and forth at least 3 times, and then wipe the back of the pharynx at least 3 times up and down, and immerse the swab head in the virus-containing preservation solution (or Use isotonic saline, tissue culture, or phosphate buffer), discard the tail, and screw the cap tightly. Sputum: Collect the coughed up sputum in a 50 mL screw-top plastic tube or sputum box containing virus preservation solution. Sputum samples were added to an equal volume of sputum digestion solution (phosphate buffered saline containing 1 g / L proteinase K), shaken and mixed, and allowed to stand for 5 minutes for digestion before nucleic acid extraction. The digested sputum sample can be used as a routine swab ...
Embodiment 3
[0031] Example 3 Detection of extracted sample nucleic acid
Prepare the amplification reaction system according to Table 2:
Table 2 Amplification reaction system configuration table
[0032] Perform RT-PCR amplification according to the following procedure: 50°C for 15min; 95°C for 5min; 95°C for 10s, 58°C for 40s, a total of 45 cycles;
Perform melting curve analysis according to the following procedure: 95°C for 10 s; 40°C for 1 min; run the melting curve analysis program from 40-85°C.
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