Automation in Molecular Diagnostics – Towards an Open Future with the BD MAX™
Table 1: Performance of the BD MAX™ Chlamydia trachomatis Assay for Urogenital Swabs in Various Commercial Transport Media and Native Urine as Compared with the Reference Assay
Amount of Specimen
e-Swabs Urine
Represented per PCR BD MAX™ Reference Assay 29 µl
12 µl 60 µl
Amplicor™ 12 µl ProbeTec™ 12 µl Viper™
12 µl Combined PCR = polymerase chain reaction.
Figure 2: Temperature Dependence of Crossing Points/Cycle Thresholds of Internal Control and Chlamydia trachomatis Amplification
22.5 25.0 27.5 30.0 32.5 35.0
20.0 51 53 55 57 59 61 63 65 67 69 71 73
Annealing temperature (ºC) CT
IC
For the optimised assay, an annealing temperature of 61 °C was chosen. CP = crossing point; CT = Chlamydia trachomatis, IC = internal control.
protocols without using the BD master mix. The open mode also allows SybrGreen assays with melting curve analysis. Extraction reagents are available for DNA or RNA, depending on the assay.
•
PCR only – For optimisation of amplification but also for assays not requiring true DNA purification (e.g. simple lysis of cultured bacteria for the detection of virulence factors), the PCR-only mode is very helpful. The assay is set up as for other thermocyclers and loaded manually into the microfluidic cartridges. The small assay volume helps to save reagents.
•
Extraction only – With BD MAX 6, the second-generation instrument it is possible to stop the process after purification of the nucleic acids, which are then available for further downstream applications.
Detection of Chlamydia trachomatis as a Model System
In order to gain experience with the new instrument, we wanted to work with an assay for which sufficient clinical specimens, positives and negatives, would be available. We therefore transferred our routinely used in-house assay for the detection of C. trachomatis to the BD MAX and validated it for various commonly used, commercially available transport systems and native urine.6
Microfluidic PCR using the same primers/probe as in the reference assay7
in a final volume of 4 µl was optimised in the PCR-only mode with regard to primer/probe concentrations as well as annealing
EUROPEAN INFECTIOUS DISEASE
Figure 3: Comparison of Crossing Points/Cycle Thresholds for Urine, e-Swabs and Cobas Amplicor Transport Medium Analysed by BD MAX™ and on LC480 (Reference Assay)
Comparison of CP in BD MAX and LC480 Amplicor
25 30 35 40 45
20 15
15 20 25 30 CP LC480
The specimen with a low cycle threshold (CP) in the reference assay but negative with BD MAX became positive upon repeating the test. Most likely, a pipetting error was responsible for the initial false negative result.
(see Figure 2) and extension temperature/time. The optimised assay comprised 50 cycles of 95 °C/10 seconds (denaturation) and 61 °C/10 seconds (annealing/elongation). Compared with our reference assay, significant adaptations were made for annealing/elongation (61 °C/10 seconds instead of 57 °C/30 seconds) and probe concentration (0.45 instead of 0.15 µM). The hot start was obsolete due to a different polymerase contained in the BD master mix. The analytical sensitivity of the assay (limit of detection – LOD) was estimated between one and 10 copies per PCR reaction based on a serially diluted quantified Quality Control for Molecular Diagnostics (QCMD) specimen and thus was comparable to the reference assay. For assay validation, 100 µl clinical specimen was used for each extraction/amplification except for urines (500 µl). The internal process control was measured in the second channel and considered positive if the crossing point (CP) was below 36.
Among the 583 clinical specimens analysed, four (0.69 %) were not sufficiently amplified and were excluded from the analysis. For the remaining 579, overall sensitivity and specificity of the BD MAX assay was 92.5 and 99.7 %, respectively (see Table 1). A comparison of crossing points between the two assays for e-Swabs, Cobas Amplicor™ and urines confirms that false negative results for BD MAX were essentially restricted to specimens with a very low target concentration (see Figure 3). At least part of this somewhat reduced sensitivity may be attributed to the fact that due to limited clinical
137 35 40 45 Urine Trend
73 µl 29 µl
100 µl 200 µl
True Positive False Positive True Negative False Negative Inhibited Sensitivity (%) Specificity (%)
10 64 90 22 49
235
0 0 0 1 0 1
112 50 90 24 48
324
0 6 5 3 5
19
0 2 1 0 1 4
100 91.4 94.7 88
90.7 92.5
100 100 100 96
100 99.7
CP
CP BD MAX
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