bioer PCR molecular

• Easy to operate: The collection process is simple and fast.

• Widely uses: Can be widely used in hospitals, scientific research institutions of saliva sample collection and transportation.

• Multiple selections: The collected samples can be used for nucleic acid extraction and virus isolation and culture at the same time for subsequent experimental operations.

• Medical material: The collector selects imported medical grade material, no heat source.

The lentivirus containing SARS-CoV-2 gene was diluted with negative saliva to a 105 copy/mL positive saliva sample，then added into the Saliva Collector（BSC92）and stored at -80℃and 4 ℃ for 35 days. After nucleic acid extraction, PCR detection was performed to determine the nucleic acid stability of SARS-CoV-2 ORF1ab gene (FAM channel) and human β-Globin internal reference gene (CY5 channel) at different storage time and temperature according to the detected CT value. The results are as follows:

Conclusion：The nucleic acids in the samples were stored at -80℃ and 4℃, and there was no significant difference in the CT values of the FAM channel and the CY5 channel detected at different time periods, indicating that the saliva collector could stably preserve the nucleic acids in the samples for one month without degradation.

Saliva or sputum specimens stored in sample collection tube for nucleic acid extraction or testing are allowed to be transported and / or stored at 2- 8°C for up to 30 days or at less than 25°C for up to 72 hours or at -70°C or below for up to 6 months.
Saliva or sputum specimens stored in sample collcetion tube for viral culture are allowed to be transported and/ or stored at less than 25°C for up to 72 hours.

Q1: The amplification efficiency is low, and there are no amplification bands in the experimental group.

A1: (1) Primers

Check if the artificially synthesized primers have degraded due to improper storage conditions; Whether the primer design is reasonable, BLAST can be used to check primer specificity or redesign primers.

(2) Template

Long term storage and repeated freezing and thawing can lead to template breakage, ring opening, or degradation. Freshly prepared DNA double strands should be used as templates; If the template GC content is too high, it will cause the double stranded DNA to be unable to open. At this time, adding PCR Enhancer (item number P021) can effectively reduce the unwinding temperature; If the template is cDNA, it is necessary to confirm the purity and integrity of the RNA used for reverse transcription.

(3) Enzymes

The enzymes used in the reaction are inactivated due to improper storage or transportation. It is recommended to replace them with new enzymes or re experiment with enzymes from another source.

(4) Amplification system

The reaction system configuration is incorrect, it is recommended to repeat the experiment.

(5) Reaction program

Check if the denaturation temperature is accurate and if the PCR instrument indicates a temperature that matches the actual temperature. If the temperature is too high, the enzyme will quickly become inactive in the first few cycles, while if the temperature is too low, the template denaturation will not be complete; The annealing temperature is not suitable. A gradient can be set for the annealing temperature to explore a suitable annealing temperature; Set annealing temperature based on primer Tm value, such as primer Tm value≥ At 72 ℃, the annealing step can be removed and the subsequent extension step (two-step PCR) can be carried out directly.

Q2: The brightness of the amplified bands is not very bright.

A2: (1) Primers

Check if the artificially synthesized primers have degraded.

(2) Template

Firstly, confirm the quality of the template. Long term storage and repeated freeze-thaw cycles can cause template breakage, ring opening, or degradation. Freshly prepared DNA double strands should be used as the template; If the template is not available, the first amplification product can be diluted in multiples and used as a template for secondary amplification.

(3) Reaction program

You can try using the Touch Down program; Extend the extension time and increase the number of cycles.

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