A Comparative Study on Visual Detection of Mycobacterium tuberculosis by Closed Tube Loop-Mediated Isothermal Amplification: Shedding Light on the Use of Eriochrome Black T

被引:14
作者
Neshani, Alireza [1 ]
Zare, Hosna [1 ]
Sadeghian, Hamid [1 ,2 ]
Safdari, Hadi [1 ,2 ]
Riahi-Zanjani, Bamdad [3 ]
Aryan, Ehsan [1 ,4 ]
机构
[1] Mashhad Univ Med Sci, Antimicrobial Resistance Res Ctr, Mashhad 9196773113, Iran
[2] Mashhad Univ Med Sci, Sch Paramed Sci, Dept Lab Sci, Mashhad 9177948964, Iran
[3] Mashhad Univ Med Sci, Med Toxicol Res Ctr, Mashhad 9177948564, Iran
[4] Mashhad Univ Med Sci, Ghaem Univ Hosp, Dept Med Microbiol, Mashhad 9176699199, Iran
关键词
LAMP; Mycobacterium tuberculosis; visual detection; EBT; calcein; HNB; DIAGNOSIS; LAMP; PCR; COMPLEX; SPUTUM; ASSAY; DNA;
D O I
10.3390/diagnostics13010155
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
Loop-mediated isothermal amplification is a promising candidate for the rapid detection of Mycobacterium tuberculosis. However, the high potential for carry-over contamination is the main obstacle to its routine use. Here, a closed tube LAMP was intended for the visual detection of Mtb to compare turbidimetric and two more favorable colorimetric methods using calcein and hydroxy naphthol blue (HNB). Additionally, a less studied dye (i.e., eriochrome black T (EBT)) was optimized in detail in the reaction for the first time. Mtb purified DNA and 30 clinical specimens were used to respectively determine the analytical and diagnostic sensitivities of each method. The turbidimetric method resulted in the best analytical sensitivity (100 fg DNA/reaction), diagnostic sensitivity and specificity (100%), and time-to-positivity of the test (15 min). However, this method is highly prone to subjective error in reading the results. Moreover, HNB-, calcein-, and EBT-LAMP could respectively detect 100 fg, 1 pg, and 1 pg DNA/reaction (the analytical sensitivities) in 30, 15, and 30 min, while the diagnostic sensitivity and specificity were respectively 93.3% and 100% for them all. Interestingly, EBT-LAMP showed the lowest potential for subjective error in reading the results. This report helps judiciously choose the most appropriate visual method, taking a step forward toward the field applicability of LAMP for the detection of Mtb, particularly in resource-limited settings.
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页数:15
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