1. What PCR machines do you validate the real time PCR assays on?
Our assays are validated on and compatible with the following real time PCR machines:
a. ABI7300/7500/7900/StepOnePlus
b. Stratagene Mx 3000P/3005P
c. Roche LightCycler 480
d. Bio-Rad IQ5/CFX96
Several assays are validated on Qiagen Rotor-Gene (Corbett) 6000/Q (see below). The assays may work on other machines, but we have not validated on machines not on this list.
2. Can your kits be used on Qiagen RotorGene (Corbett) 6000/Q (i.e. RGQ)?
Currently only the following assays have been validated on and compatible with this PCR machine:
a. EGFR Three Mutations Detection Kit (L858R, E746_A750del (1), E746_A750del (2))
b. KRAS Seven Mutations Detection Kit
c. BRAF V600E Mutation Detection Kit
d. EGFR 29 Mutations Detection Kit will be available in the near future
e. EML4-ALK Fusion Gene Detection Kit for Rotor-Gene Q
Other kits may work on Qiagen RotorGene 6000/Q, but we have not validated them yet.
3. Why does the PCR protocol have two rounds of amplification?
In the first round of amplification, the annealing temperature is relatively high. This high temperature makes for high specificity, low efficiency amplification, resulting in enrichment for the target sequence. In the second stage, the temperature is lower and primers that were unable to anneal in the first stage can now bind and amplify tagged DNA generated in the initial round of PCR. This second stage is also highly specific, amplifying only target sequences, but is also very efficient. The PCR tube is not opened to introduce the second set of primers - the two stage amplification uses different temperatures to take advantage of the two primer sets.
4. Can you give me a general description of the specific technology that is used in the kits?
We use our patented ADx-ARMS real-time PCR technology to detect the mutations in the target DNA. Target DNA is amplified with mutation-specific PCR primers, and the mutant amplicons are detected with a novel fluorescent probe.
5. What’s the advantage of your PCR technology compared to other methods?
Our advantage is as follows:
a. Simple procedure: only one step required for sample application, which keeps the contamination at a minimum level; results obtained within 90 minutes.
b. High sensitivity and accuracy: can detect 1% mutant DNA in a background of wild type genomic DNA, while ensuring that false negatives are minimized.
c. Compatible with multiple real time PCR machines.
d. External and Internal controls; positive and negative controls.
e. EGFR, BRAF and KRAS kits are CE marked for IVD use in Europe and SFDA approved for clinical use in China.
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Below please find the comparison chart (from AstraZeneca):
6. Whom should I contact to discuss quality assessment and result analysis?
Please choose either of the following methods:
a. Please call us at 86-592-6806835; if you’re in US, please call 650-954-1055.
b. Please send emails to Sales@amoydx.com and we will forward your questions to our scientists for immediate analysis. If you have a question about the PCR output, please include the data file in your email.
7. Why do the Ct values of the internal HEX control reactions vary for the same sample?
The HEX/VIC signal is based on amplification of normal human genomic DNA. The purpose of HEX/VIC control is to monitor the addition of DNA (ie, a loading control). Because there may be some competition between the FAM (mutation) and HEX signals, we adjust the amount of probe and primers for the HEX in each well to guarantee the FAM signal´s sensitivity and specificity. Each reaction system for HEX in the different wells is distinctive. This results in different amplification efficiency of HEX amplicons among the sample wells, and thus variant Ct values for wells even with the same amount of sample DNA.
"Pre-loaded" kits have the buffer, primers, probes, and nucleotides pre-loaded into the PCR strips. The user just adds the sample DNA and Taq enzyme (included in the kit). The EGFR and KRAS kits are also available "in-bulk", which means the user pipettes the reagents into the PCR tubes.
9. Does the Light Cycler 480 PCR machine require the use of an adapter for PCR strips?
Yes, the adapter is available from your local Roche dealer, or can be ordered from the company Bioplastics.
10. How are the kits delivered?
We ship on "blue ice" packed with foam and carton boxes, and use TNT or Fedex for most of our shipping but other carriers may be more suited for specific countries. The customers can also use their own forwarders and choose to pay the shipping fee by collect.
11. What’s the shelf life of your kits?
a. EGFR, BRAF and KRAS kits: 6 months shelf life
b. Other kits (such as EML4-ALK, JAK2, PIK3CA): 8 months shelf life
12. What’s the size of your kit box and carton box?
EGFR and KRAS kit box: 159*115*53mm (6.3”x4.5”x2.1”)
BRAF and others: 80*60*53mm (3.1”x2.4”x2.0”)
Carton box: 420x340x340mm (16.5”x13.4”x13.4”)