Abstract：
BACKGROUND: Currently for diagnosing Mycobacterium tuberculosis and its drug resistance, two sputum samples are required. One of them is subjected to TrueNat™ and if positive the other sample is subjected to line probe assay (LPA). This study was done to evaluate whether TrueNat extracted DNA can be directly used for performing LPA in a diagnostic laboratory setting to decrease patient turn-around time.
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.
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.
摘要：
背景：目前用于诊断结核分枝杆菌及其耐药性，需要两份痰样本。将它们中的一个进行TrueNat™,并且如果为阳性,则将另一个样品进行线探针测定(LPA)。进行这项研究是为了评估TrueNat提取的DNA是否可以直接用于在诊断实验室环境中进行LPA以减少患者周转时间。
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。
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。
