Objective To evaluate the performance of the detection of Mycobacterium tuberculosis(MTB)nucleic acids on the CFX Opus 96 Dx real-time fluorescence PCR(RT-PCR)analyzer to determine its clinical applicability.Methods 20 clinical sputum samples were collected,and MTB bacterial suspensions were serially diluted to prepare the samples.After extraction of nucleic acids using thermal lysis,MTB DNA was amplified by real-time fluorescent PCR(RT-PCR)using the MTB nucleic acid detection reagent on the CFX Opus 96 Dx real-time fluorescence PCR analyzer to evaluate the performance of the method,including the limit of detection(LOD),precision,cross-reactivity,anti-interference ability,personnel and inter-instrument comparison,and methodological compliance rate.Results The minimum detection limit of the CFX Opus 96 Dx real-time fluorescence PCR analyzer for detecting MTB DNA was 1×102 bacteria/ml,which aligns with the requirements of the reagent specification.The intra-batch coefficient of variation(CV)was 1.04%,1.61%and 1.29%for 3 repetitions of 5×102 bacteria/ml samples.The intra-batch CVs were 0.92%,0.74%and 0.59%for 3 repetitions of 5×103 bacteria/ml samples,which were all<5%.Common respiratory pathogens such as Staphylococcus aureus,Staphylococcus epidermidis,Pseudomonas aeruginosa,Klebsiella pneumoniae,Nocardia,Proteus mirabilis,Legionella pneumophila,Pneumocystis japonicus,Influenza A virus,Influenza B virus,Mycoplasma pneumoniae and SARS-CoV-2 do not cross-react with MTB.There was no statistically significant difference in the change in cycle threshold(Ct)values before and after the addition of the interfering substances Hemoglobin,Mucin,Rifampicin,Isoniazid,Amoxicillin and Levofloxacin to the MTB-positive specimens(all P>0.05).Comparison CVs between the two operators and all three PCR instruments were less than 5 percent.The positive compliance rate,negative compliance rate and total compliance rate of MTB DNA detected by the ABI 7500 real-time PCR analyzer and the CFXOpus 96 Dx real-time PCR analyzer were 100%.Conclusion The LoD,precision,cross-reactivity,anti-interference ability,personnel and inter-instrument comparison,and methodological compliance rate of the CFX Opus 96 Dx real-time fluorescence PCR analyzer for the detection of MTB DNA are all in line with the requirements of clinical molecular biology testing,which can provide a reliable basis for clinical testing.

Objective To evaluate the performance of the detection of Mycobacterium tuberculosis(MTB)nucleic acids on the CFX Opus 96 Dx real-time fluorescence PCR(RT-PCR)analyzer to determine its clinical applicability.Methods 20 clinical sputum samples were collected,and MTB bacterial suspensions were serially diluted to prepare the samples.After extraction of nucleic acids using thermal lysis,MTB DNA was amplified by real-time fluorescent PCR(RT-PCR)using the MTB nucleic acid detection reagent on the CFX Opus 96 Dx real-time fluorescence PCR analyzer to evaluate the performance of the method,including the limit of detection(LOD),precision,cross-reactivity,anti-interference ability,personnel and inter-instrument comparison,and methodological compliance rate.Results The minimum detection limit of the CFX Opus 96 Dx real-time fluorescence PCR analyzer for detecting MTB DNA was 1×102 bacteria/ml,which aligns with the requirements of the reagent specification.The intra-batch coefficient of variation(CV)was 1.04%,1.61%and 1.29%for 3 repetitions of 5×102 bacteria/ml samples.The intra-batch CVs were 0.92%,0.74%and 0.59%for 3 repetitions of 5×103 bacteria/ml samples,which were all<5%.Common respiratory pathogens such as Staphylococcus aureus,Staphylococcus epidermidis,Pseudomonas aeruginosa,Klebsiella pneumoniae,Nocardia,Proteus mirabilis,Legionella pneumophila,Pneumocystis japonicus,Influenza A virus,Influenza B virus,Mycoplasma pneumoniae and SARS-CoV-2 do not cross-react with MTB.There was no statistically significant difference in the change in cycle threshold(Ct)values before and after the addition of the interfering substances Hemoglobin,Mucin,Rifampicin,Isoniazid,Amoxicillin and Levofloxacin to the MTB-positive specimens(all P>0.05).Comparison CVs between the two operators and all three PCR instruments were less than 5 percent.The positive compliance rate,negative compliance rate and total compliance rate of MTB DNA detected by the ABI 7500 real-time PCR analyzer and the CFXOpus 96 Dx real-time PCR analyzer were 100%.Conclusion The LoD,precision,cross-reactivity,anti-interference ability,personnel and inter-instrument comparison,and methodological compliance rate of the CFX Opus 96 Dx real-time fluorescence PCR analyzer for the detection of MTB DNA are all in line with the requirements of clinical molecular biology testing,which can provide a reliable basis for clinical testing.