According to the Technical Guidelines for the Individual Treatment and Detection of Cancer (Trial), when collecting peripheral blood to extract plasma free DNA for detection, a one-time closed EDTA anticoagulant Vacutainer should be used to collect 6~10ml of whole blood, refrigerate and transport it, separate plasma within 6 hours, extract free DNA, store it in a refrigerator at -80 ℃, and avoid repeated freezing and thawing. If peripheral blood needs to be transported for a long time, it is recommended to use commercial free DNA sample storage tubes. Under normal temperature conditions, cfDNA can be stably stored in whole blood for 7 days.

The most widely used commercial free DNA sample storage tube is the Street ® Cell Free DNA ™ (Street BCTs) blood collection vessels. Recently, some domestic companies such as Kangwei Century and Mei'an Biology have also launched free DNA sample preservation tubes.

The following is a quotation of Chen Yinqing's (official account: the possibility of digital PCR) "The more detailed, the more critical - sample preservation and nucleic acid extraction" ® Cell Free DNA ™ The advantages of (Street BCTs) blood collection compared to traditional EDTA blood collection in peripheral blood preservation.

Optimization of sample preservation

In this study, a total of 46 Rh negative pregnant women (28 to 30 weeks of pregnancy) participated in the determination of fetal gender and Rh blood type through cffDNA testing. The specific detection was carried out using micro drop digital PCR and compared with the results of fluorescence quantitative PCR.

In the step of blood sample collection, the article set up a comparative experiment: traditional EDTA blood collection and Street ® Cell Free DNA ™ (Street BCTs) blood collection vessels. The two types of blood samples preserved from blood collection vessels were purified using QIAgen's QIAamp Circulating Nuclear Acid kit, and then subjected to ddPCR and qPCR detection, respectively. StreckBCTs blood collection tubes contain a special component (cell stabilizing agent, editor's note), which can inhibit the lysis of mother cells, thus increasing the concentration of cffDNA in plasma. The results showed that the cffDNA content of the blood samples stored in the Street BCTs collection vessel after nucleic acid extraction was 4-24%, while the cffDNA content corresponding to the EDTA collection vessel was 0.1-2%.

By analyzing the gene content of SRY, TSPY1, RHD5, and RHD7 through digital PCR, it was found that the average content of cffDNA in the blood samples preserved by Street BCTs was 9-16%, significantly higher than that of the EDTA treated blood samples. The average content of cffDNA in the latter was only (0.5-1%) (P<0.001).

The internal reference genes Xp22.3 and EIF2C1 in the blood are not only derived from the fetus, but also from the mother. Quantitative analysis using digital PCR showed that the content of EDTA preserved samples was significantly higher than that of Street BCTs preserved samples (P<0.001). The content of the former is 548.04 and 869.25 copies="">
As shown in Figure B below, due to the release of a large amount of maternal DNA, the absolute copy numbers of internal reference genes (Xp22.3 and EIF2C1) in EDTA blood samples were significantly higher than those in Street BCTs, resulting in a decrease in cffDNA concentration. As shown in Figure A, the content of SRY, TSPY1, and RHD genes derived from fetuses, as well as the proportion of EDTA blood samples collected for preservation, is much lower than that of Street BCTs blood samples.

Cell stabilizing agent

Maintaining the initial concentration of free DNA in plasma is crucial for subsequent research. The free DNA sample storage tube contains a cell stabilizer that can reduce the release of gDNA into plasma. Traditional cell stabilizers include formaldehyde, Glutaraldehyde, etc. Aldehyde cell stabilizers can crosslink DNA and DNA Protein, thus damaging the double helix structure of DNA.

The effect of residual cell stabilizers on subsequent enzyme reactions

Street ® Cell Free DNA ™ Formaldehyde and Glutaraldehyde cell stabilizers were not used in the blood collection vessel, and there were literatures to detect the street ® Cell Free DNA ™ The blood collection vessel is formally dehydrated free. However, relevant literature, including the specifications of the Streck BCTs, pointed out that the plasma collected with this blood collection vessel needs to be heated with Proteinase K in order to obtain the best effect. That is to say, no matter what kind of cell stabilizer, the traditional lysis cleaning method will have residues, which will affect the subsequent enzyme reaction. This effect can only be completely removed after Proteinase K treatment.

Automatic free DNA extraction extraction

At present, in the field of non-invasive prenatal screening, third-party testing institutions face high-throughput samples, and manual operations are not feasible in terms of quality control or sample quantity. So for the nucleic acid extraction step, the simpler the better. We will not discuss the feasibility of automating the centrifugal column method, as magnetic bead purification alone has great advantages in terms of flux.

On the other hand, considering the disadvantages of EDTA preservation tubes compared to free DNA preservation tubes, more and more NIPT testing institutions have gradually adopted free DNA preservation tubes to replace EDTA preservation tubes. (Street BCTs are certainly the top choice, but in terms of price, they are not ideal. More and more excellent domestic preservation tube manufacturers are committed to solving this problem, and I believe that brand localization is also the future trend of the entire biological industry.)

According to the optimal treatment method recommended by Streck, Proteinase K heating treatment is required. However, in actual operation, the testing company will consider whether the additional step will affect the automation of the extraction process, but more concerned is whether the aerosol caused by heating will have serious consequences of cross contamination on the test results of clinical samples.

So here is a magnetic bead free DNA extraction kit BioBase UPure Circulating DNA Plus Kit (For Street Cell free DNA BCT)

The BioBase UPure Circulating DNA Kit, the first generation cfDNA extraction kit, has only three simple components and does not require heating, perfectly matching various external built-in magnetic field automated nucleic acid extraction instruments. Widely used in well-known testing institutions in China, such as Huada, Annuoyuda, Da'an, Boaomuhua, etc.

The second generation of reagent kit, BioBase UPure Circulating DNA Plus Kit (For Streck Cell free DNA BCT), which is aimed at extracting plasma samples from Streck cell free DNA BCT and domestic free DNA preservation tubes, has solved the problem that Proteinase K heating is required to remove the effects of cell stabilizers with a unique reagent formula. It still adheres to the consistent characteristics of BioBase reagent kit, with simple components, few operating steps and no heating, No preprocessing is required. Users of the first generation of products can replace the entire line without even needing to modify automated operating procedures.

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