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Application of hyperbranched rolling circle amplification for direct detection of mycobacterium tuberculosis in clinical sputum specimens.

Liu Y, Guo YL, Jiang GL, Zhou SJ, Sun Q, Chen X, Chang XJ, Xing AY, Du FJ, Jia HY, Zhang ZD - PLoS ONE (2013)

Bottom Line: The results of all MTC strains were positive in contrast to the NTM specimens which were all negative.Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture.Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology for Tuberculosis, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital Medical University, Beijing, China.

ABSTRACT

Background: Global tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens.

Methods: A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb.

Results: The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72).

Conclusions: Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.

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Detection limits of HRCA.Agarose gel analysis of HRCA products. Line1 (the left most) is 100 bp DNA marker. Line 2 to 9 (from left to right), 10 µ of the HRCA products were analyzed b 2% agarose gel electrophoresis followed by ethidium bromide staining. The specimen detected by HRCA: line 2 to line 5 is H37Rv purified DNA of 740 fM, 74 fM,7.4 fM, 740 aM; line 6 to 9 is H37Rv culture suspension of 2×105 cfu/ml, 2×104 cfu/ml, 2×103 cfu/ml, 2×102 cfu/ml. Because no bands were seen in the 74 aM and 2×101 cfu/ml samples, the sensitivity of HRCA in detection of purified DNA and culture suspension is 740 aM and 2×102 cfu/ml, respectively.
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pone-0064583-g001: Detection limits of HRCA.Agarose gel analysis of HRCA products. Line1 (the left most) is 100 bp DNA marker. Line 2 to 9 (from left to right), 10 µ of the HRCA products were analyzed b 2% agarose gel electrophoresis followed by ethidium bromide staining. The specimen detected by HRCA: line 2 to line 5 is H37Rv purified DNA of 740 fM, 74 fM,7.4 fM, 740 aM; line 6 to 9 is H37Rv culture suspension of 2×105 cfu/ml, 2×104 cfu/ml, 2×103 cfu/ml, 2×102 cfu/ml. Because no bands were seen in the 74 aM and 2×101 cfu/ml samples, the sensitivity of HRCA in detection of purified DNA and culture suspension is 740 aM and 2×102 cfu/ml, respectively.

Mentions: In order to investigate the sensitivity of HRCA for purified DNA, which was extracted and purified from H37Rv culture using QIAamp DNA Mini Kit (Qiazen, USA), the purified DNA was diluted by a series of 1∶10 dilutions from 7.4 pM to 7.4 aM and then tested by HRCA and qRT-PCR. The sensitivity of the HRCA and qRT-PCR for purified DNA was 740 aM (Fig. 1) and 74 aM (Fig. 2), respectively.


Application of hyperbranched rolling circle amplification for direct detection of mycobacterium tuberculosis in clinical sputum specimens.

Liu Y, Guo YL, Jiang GL, Zhou SJ, Sun Q, Chen X, Chang XJ, Xing AY, Du FJ, Jia HY, Zhang ZD - PLoS ONE (2013)

Detection limits of HRCA.Agarose gel analysis of HRCA products. Line1 (the left most) is 100 bp DNA marker. Line 2 to 9 (from left to right), 10 µ of the HRCA products were analyzed b 2% agarose gel electrophoresis followed by ethidium bromide staining. The specimen detected by HRCA: line 2 to line 5 is H37Rv purified DNA of 740 fM, 74 fM,7.4 fM, 740 aM; line 6 to 9 is H37Rv culture suspension of 2×105 cfu/ml, 2×104 cfu/ml, 2×103 cfu/ml, 2×102 cfu/ml. Because no bands were seen in the 74 aM and 2×101 cfu/ml samples, the sensitivity of HRCA in detection of purified DNA and culture suspension is 740 aM and 2×102 cfu/ml, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672175&req=5

pone-0064583-g001: Detection limits of HRCA.Agarose gel analysis of HRCA products. Line1 (the left most) is 100 bp DNA marker. Line 2 to 9 (from left to right), 10 µ of the HRCA products were analyzed b 2% agarose gel electrophoresis followed by ethidium bromide staining. The specimen detected by HRCA: line 2 to line 5 is H37Rv purified DNA of 740 fM, 74 fM,7.4 fM, 740 aM; line 6 to 9 is H37Rv culture suspension of 2×105 cfu/ml, 2×104 cfu/ml, 2×103 cfu/ml, 2×102 cfu/ml. Because no bands were seen in the 74 aM and 2×101 cfu/ml samples, the sensitivity of HRCA in detection of purified DNA and culture suspension is 740 aM and 2×102 cfu/ml, respectively.
Mentions: In order to investigate the sensitivity of HRCA for purified DNA, which was extracted and purified from H37Rv culture using QIAamp DNA Mini Kit (Qiazen, USA), the purified DNA was diluted by a series of 1∶10 dilutions from 7.4 pM to 7.4 aM and then tested by HRCA and qRT-PCR. The sensitivity of the HRCA and qRT-PCR for purified DNA was 740 aM (Fig. 1) and 74 aM (Fig. 2), respectively.

Bottom Line: The results of all MTC strains were positive in contrast to the NTM specimens which were all negative.Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture.Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology for Tuberculosis, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital Medical University, Beijing, China.

ABSTRACT

Background: Global tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens.

Methods: A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb.

Results: The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72).

Conclusions: Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.

Show MeSH
Related in: MedlinePlus