Qihengxing blood sample direct PCR/qPCR solution

In clinical testing, blood is one of the most important test samples. With the rapid development and popularization of molecular detection technology, genetic testing technology is now widely used in disease detection or early disease screening. Blood samples contain a large amount of autologous or pathogen nucleic acid. By testing DNA/RNA in the blood, a large amount of information can be obtained for disease screening and health management, such as genetic diseases, early disease screening (such as early tumor screening), infectious disease detection or forensic identification.

In the field of molecular diagnosis, commonly used detection methods include PCR, qPCR, and next-generation sequencing. The current conventional detection method is to extract and purify nucleic acids first, and then perform downstream detection on the extracted nucleic acids. Taking DNA extraction as an example, the extraction time takes 1-2 hours, the PCR/qPCR detection time is about 1 hour, and the entire experimental process takes about 3 hours.

In order to improve the efficiency of blood testing, a detection method that directly performs PCR/qPCR on blood after pretreatment has emerged. However, this method is limited in application scenarios due to factors such as excessive sample pretreatment, low amplification efficiency, and poor anti-inhibition.

Beijing Qihengxing Biotechnology has achieved efficient PCR/qPCR detection of a variety of blood samples after simple lysis by constructing and screening genetically engineered enzymes and optimizing amplification buffers. The entire experimental process is shortened to 1 hour, and it also has excellent anti-interference capabilities and stable amplification efficiency.

Direct PCR in blood Premix ( StarLighter Blood Direct PCR Mix ）

StarLighter Blood Direct PCR Mix uses a unique PCR buffer system that can effectively reduce the impact of inhibitors in the blood on amplification. It can perform direct PCR using whole blood samples as templates without DNA purification or sample processing. It is suitable for EDTA anticoagulated blood samples under different storage conditions and can be used for single or multiplex blood direct amplification.

Product advantages and experimental data

1. Suitable for multiple amplification, compatible with templates with different GC% contents, good amplification uniformity, and a wide range of blood input volume.

Fig2.1 The blood direct amplification kits from Qihengxing and supplier T were used to perform direct amplification with 6 primers on fresh EDTA anticoagulated blood in different proportions, namely 5%, 10%, 15%, 20%, 25% and 30%. The amplified fragment ranged from 200-800bp and the GC% ranged from 35% to 77%. After 35 cycles of amplification, the PCR products were detected by agarose gel electrophoresis.
The results showed that the Qihengxing blood direct amplification kit was superior to that of supplier T in terms of product quantity and amplification uniformity.

Fig 2.2 Results of direct amplification of EDTA anticoagulated blood stored at room temperature

For EDTA blood samples stored at different conditions such as room temperature, 4°C, and -20°C, StarLighter Blood Direct PCR Mix can accurately amplify the products of six items.

Fig 2.3 Results of direct amplification of EDTA anticoagulated blood stored at 4℃ and -20℃

PART TWO Simple blood lysis direct expansion qPCR Reagents box (StarLighter Probe qPCR Kit for Blood)

StarLighter Probe qPCR Kit for Blood It is suitable for various types of blood samples and does not require tedious nucleic acid extraction and purification. It only requires simple lysis of the blood sample and taking the supernatant for qPCR amplification. It is easy to operate and can reduce the risk of contamination. The test results are consistent with those of the pure DNA product after nucleic acid extraction as a template.

Product advantages and experimental data

1. Fast operation, from blood sample to complete genotyping/quantification analysis , Solve big problems in 1 hour .

Fig 3.1 StarLighter Probe qPCR Kit for Blood operation flow chart

2. SNP typing is accurate, Compatible with various types of samples : Fresh blood sample A and refrigerated (stored at 4°C for 2 months) blood sample A with known SNP genotyping For three gene loci Test, results Both Sanger sequencing is completely consistent .

Fig 3.2 Detection results of different SNP sites after lysis and release of fresh blood sample A. Blood was lysed and centrifuged to obtain the supernatant, and 4 μL of the supernatant was added to the 20 μL reaction system as a template for qPCR. The detection results were consistent with the Sanger sequencing results.

Fig 3.3 Detection results of different SNP sites after lysis and release of stored blood sample A. Blood was lysed and centrifuged to obtain the supernatant, and 4 μL of the supernatant was added to the 20 μL reaction system as a template for qPCR. The detection results were consistent with the Sanger sequencing results.

3. Test results and carry After purification, qPCR Consistency : For the same sample, StarLighter Probe qPCR Kit for Blood Reagent test kit and tradition Blood liquid Genomic DNA Extraction Kit Then qPCR and other two methods were used to Specific gene loci The experimental results show that two Methods of detection Ct value Very consistent with the fluorescence intensity .

Fig 3.4 Comparison of qPCR data after extraction using the Qihengxing StarLighter Probe qPCR Kit for Blood and the traditional blood nucleic acid extraction kit. The thick line is the qPCR result after nucleic acid release solution treatment, and the thin line is the qPCR result after whole blood genomic DNA extraction and purification. The blue is the FAM channel, the orange is the Rox channel (internal reference), and the green is the Vic channel.

From technological breakthroughs to scene implementation, Qihengxing Bio has integrated multi-platform product development experience such as sample pre-treatment, core enzyme development, and buffer system optimization, and has created a full-process solution with simple lysis and direct amplification of blood DNA as the core. Whether it is the high purity guarantee of automated/manual whole blood extraction, the minimalist breakthrough of direct amplification PCR, or the high efficiency and accuracy of rapid lysis qPCR, each technology embodies a deep insight into the pain points of the industry and innovative practices. In the future, Qihengxing will continue to work hard to promote the expansion of blood gene testing technology to deeper and broader dimensions, promote the optimization of the entire process from sample processing to precise analysis, and provide product application support for more application scenarios.