METHODS: Total 45 smear positive sputum samples were subjected to TrueNat™ MTB detection and first and second line (FL and SL) LPA testing in parallel. DNA extracted by Trueprep® Cartridge was also tested by LPA and results were compared. Further, TrueNat extracted DNA from 20 samples was divided into 6 aliquots each, two of which were stored at 4 °C, 37 °C and 55 °C (under humidification) each. One aliquot from stored DNA at each temperature was used for FL & SL LPA on day three and the other on day eight. The blots thus obtained were compared with those of conventional LPA at day 1.
RESULTS: For FL-LPA, TrueNat extracted DNA gave valid results for all 45 (100%) samples but conventionally extracted DNA could give results for 44 (97.8%) samples. Likewise, for SL-LPA, valid results were obtained for 40 (88.9%) and 35 (77.8%) samples respectively using TrueNat extracted DNA and conventionally extracted DNA respectively. All samples with invalid LPA results had Ct values ≥ 28 by TrueNat PCR. LPA results were obtained for all the 20 samples using stored DNA at all temperatures and duration.
CONCLUSIONS: TrueNat extracted DNA can be used for performing LPA under field conditions for selected samples.
方法:对总共45个涂片阳性痰样品进行TrueNat™MTB检测以及一线和二线(FL和SL)LPA平行检测。通过Trueprep®Cartridge提取的DNA也通过LPA测试并比较结果。Further,TrueNat从20个样本中提取的DNA被分成6个等分试样,其中两个储存在4°C,各37°C和55°C(在加湿下)。每个温度下来自储存的DNA的一个等分试样在第三天用于FL&SLLPA,另一个在第八天用于FL&SLLPA。在第1天将由此获得的印迹与常规LPA的印迹进行比较。
结果:对于FL-LPA,TrueNat提取的DNA对所有45个(100%)样品给出有效结果,但是常规提取的DNA可以对44个(97.8%)样品给出结果。同样,对于SL-LPA,分别使用TrueNat提取的DNA和常规提取的DNA对40份(88.9%)和35份(77.8%)样品分别获得了有效结果。通过TrueNatPCR,具有无效LPA结果的所有样品的Ct值≥28。在所有温度和持续时间下使用储存的DNA获得所有20个样品的LPA结果。
结论:TrueNat提取的DNA可用于在田间条件下对所选样品进行LPA。