Revolutionize your pipetting Picus
Figure 3 – Amplification curves for Mycoplasma fermentans samples with and without previous Vivaspin concentration step, including coating buffer.
• Pipetting of 50 μL of the master mix plus 50 μL of the sample or positive or negative control material (e.g., elution buffer, PCR water, or 10 mM tris buffer) into 200-μL PCR tubes and insertion into the preprogrammed real-time thermoycler.
The results are illustrated in Tables 1 and 2 and Figure 3. The Ct (cycle threshold) value of the concentrated samples was always compared with samples that were not concentrated. The Ct value is the amplification cycle at which a certain fluorescence threshold value is exceeded. Table 1 lists the individual Ct values. Table 2 shows the average delta (Δ) Ct value from Table 1 and the Ct difference, which results from using or not using the coating buffer. The ΔCt value is determined as follows: ΔCt = Ct (sample without Vivaspin concentration) – Ct (concentrated sample).
With the concentration in combination with the coating buffer, a ΔCt value of >7 could be achieved for Mycoplasma fermentans. When the coating buffer was not used (individual values are not listed) a lower ΔCt value of ~6 was recorded, although it was possible to take benefit from the shorter centrifugation times of the Vivaspin units. However, this still is a considerable increase in sensitivity.
Conclusion Parameters such as sensitivity and time savings are essential for the inves- tigation of mycoplasma contamination. This is accomplished with the PCR Kit in combination with the concentration step described above. By using Vivaspin 20, a ΔCt improvement >7 can be achieved, which corresponds to an increase in sensitivity of approx. 2 log levels. This makes the Microsart AMP Mycoplasma Detection Kit and Vivaspin 20 a cost-effective, practical, and rapid test for mycoplasma.
The authors are with Sartorius AG, August-Spindler-Strasse 11, 37079 Göt- tingen, Germany; tel.: +49 551 3080; fax: +49 551 308 3289; e-mail: info@
sartorius.com;
www.sartorius.com
AMERICAN LABORATORY • 11 • JUNE/JULY 2014
www.sartorius.com/picus
Biohitfamily
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64