How to choose the right blood collection tube for your sample?
How to select inactivated and non-inactivated virus preservation solutions?
How can I effectively extract nucleic acids from viscous sputum samples?
At the end of 2019, a new coronavirus pneumonia raged in China. Due to its high contagiousness, the country paid great attention to this outbreak and organized national efforts to prevent and control the epidemic. The research community isolated the novel coronavirus strain and deciphered its nucleic acid sequence in a short period of time. The new coronavirus nucleic acid detection technology became popular and entered the public eye, opening the era of pathogenic nucleic acid detection.
Pathogenic nucleic acid testing includes sample collection, nucleic acid extraction, and nucleic acid amplification. All can have a decisive impact on the final result. The timing and location of sample collection, sample handling and preservation, the efficiency of nucleic acid extraction, and the sensitivity of amplification reagents all affect the interpretation of the final results. I am sure you are familiar with nucleic acid extraction and PCR amplification. How much do you know about sample collection and pretreatment? Next, let me take you through.
Pathogenic nucleic acid detection in the clinical common sample types are tissue, blood, swabs, sputum, etc., some of the samples are viscous, direct extraction effect is not good. I often encounter partners ask about the handling of these samples, here I made a summary of the summary, I hope it will be helpful to you.
Tissue samples
Block tissue samples cannot be fully lysed in a short period of time and generally require sample pre-treatment. Fresh tissue samples are washed twice with physiological saline, and then the cleaned tissue blocks are made into homogenate by glass sand grinding or tissue grinder, added to physiological saline or PBS for vigorous shaking and mixing, centrifuged, and the supernatant is taken for nucleic acid extraction.
Blood samples
It is sufficient to use vacuum blood collection tubes containing anticoagulants for preservation. However, there are various types of anticoagulants in blood collection tubes, and the type of anticoagulant can directly affect the results of subsequent tests. I consult professional books, it is recommended to use EDTA or sodium citrate anticoagulation tube, not recommended to use heparin anticoagulation tube (because heparin is difficult to remove in the later nucleic acid extraction process, and heparin has a strong inhibitory effect on PCR reaction, so it is not recommended to use heparin anticoagulation tube). After collecting the blood, invert and mix for later use.
Serum or plasma samples
The supernatant separated from whole blood without anticoagulant is serum, and the supernatant separated from whole blood with anticoagulant is plasma. After whole blood is taken, it is rested for 1-2 h and placed in a centrifuge for 15 min at 3000 rpm to obtain serum or plasma.
Swab samples
Swab samples are usually stored in virus preservation tubes. Virus preservation tubes are divided into inactivated virus preservation tubes and non-inactivated virus preservation tubes depending on the composition of the preservation solution. Non-inactivated virus preservation solution is a modified virus maintenance solution based on transport medium, which can retain virus activity and is mostly used for isolation and identification of virus strains and virus culture. Inactivated virus preservation solution is a modified preservation solution based on nucleic acid extraction lysate, which can lyse inactivated viruses while protecting viral nucleic acids from degradation, and is usually used for pathogenic nucleic acid detection. Sampling swabs directly contact the sampling site, the material of the swab will also have an impact on the subsequent test results, of which velvet swabs are widely used because of their good water absorption and more conducive to the release of viral properties. After sampling, the swab is well shaken and mixed in the preservation solution, and the supernatant is taken for nucleic acid extraction.
Sputum samples
Sputum contains a large amount of mucin and impurities and is relatively viscous, which makes direct nucleic acid extraction ineffective, so sputum pretreatment is performed. The sputum is liquefied with NaOH or denaturant, and then the precipitate is centrifuged for nucleic acid extraction.
