Quantitation of pulmonary fungal burden in <i>Paracoccidioides brasiliensis-infected</i> mice by real-time PCR

Although colony-forming unit (CFU) counting is widely used to quantify fungal load in tissue from animal experimentally infected with Paracoccidioides brasiliensis, several technical disadvantages have been described. Here we developed highly accurate quantitative PCR (qPCR) assays to determine the relative P brasiliensis load in lungs from infected mice. SYBR Green- and TaqMan-based assays using primers and probe for the 43-kDa glycoprotein (gp43) gene detected as little as 270 gene copies (about 2 fg of DNA) per reaction. Although qPCR assays cannot distinguish between living and dead yeasts, we found a highly positive linear correlation between CFU and qPCR.

KEYWORDS:
Paracoccidioides brasiliensis; Paracoccidioidomycosis; Quantitative PCR; gp43 gene; Experimental infection; Real-Time PCR

Authorship

Marcelo Vieira Costa

Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, São Paulo, BrazilUniversidade de São PauloBrazilRibeirão Preto, São Paulo, BrazilUniversidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, São Paulo, Brazil

Taise Natali Landgraf

Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Programa de Pós-Graduação em Imunologia Básica e Aplicada, Ribeirão Preto, São Paulo, BrazilUniversidade de São PauloBrazilRibeirão Preto, São Paulo, BrazilUniversidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Programa de Pós-Graduação em Imunologia Básica e Aplicada, Ribeirão Preto, São Paulo, Brazil

http://orcid.org/0000-0003-2084-7340

Priscila C. Corrêa

Igor Emiliano Lemos Souza

Fabrício Freitas Fernandes

Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Biologia Celular e Molecular, Ribeirão Preto, São Paulo, BrazilUniversidade de São PauloBrazilRibeirão Preto, São Paulo, BrazilUniversidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Biologia Celular e Molecular, Ribeirão Preto, São Paulo, Brazil

Ademilson Panunto-Castelo Correspondence to: Ademilson Panunto-Castelo Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Av. Bandeirantes, 3900, Monte Alegre, CEP 14040-901, Ribeirão Preto, SP, Brazil E-mail:apcastelo@usp.br

http://orcid.org/0000-0003-0148-0133

Correspondence to: Ademilson Panunto-Castelo Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Av. Bandeirantes, 3900, Monte Alegre, CEP 14040-901, Ribeirão Preto, SP, Brazil E-mail:apcastelo@usp.br

CONFLICT OF INTERESTS

The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the manuscript.

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Figures | Formulas

Figures (2)
Formulas (1)

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Figure 1
Standard curves for qPCR - A) Correlation between the amount of genomic DNA from P. brasiliensis (log) and Cq. Four amounts of genomic DNA from P brasiliensis (ng) were submitted to qPCR using GP43 sequence-specific primers, which amplify 137 nt, and the DNA intercalating dye SYBR Green I. Cq (quantification cycle) consists of the number of cycles where there is the intersection of the cut line in the exponential phase of the amplification curve; B and C) Standard curves were constructed by plotting quantification cycle values (Cq) against an estimated DNA copy number to GP43 gene. Serial 10-fold dilutions of the plasmid pET28a-GP43 ranging from 2 ng to 2 fg (2.7 × 10⁸8. Buitrago MJ, Merino P, Puente S, Gomez-Lopez A, Arribi A, Zancope-Oliveira RM, et al. Utility of real-time PCR for the detection of Paracoccidioides brasiliensis DNA in the diagnosis of imported paracoccidioidomycosis. Med Mycol. 2009;47:879-82. to 2.7 × 10²2. Shikanai-Yasuda MA, Telles Filho FQ, Mendes RP, Colombo AL, Moretti ML. Consenso em paracoccidioidomicose. Rev Soc Bras Med Trop. 2006;39:297-310. copy number) were used as template in a qPCR run to construct the standard curves. The reactions were performed with the primers described above and either the dye SYBR Green I; B) or an allele-specific fluorogenic probe (TaqMan); C). All reactions were preformed in a CFX96 Touch Real-Time PCR Detection System. Error bars represent the standard deviation of duplicate PCR reactions.

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Figure 2
The relationship between CFU per gram of lung and copy number of the GP43 gene obtained from the lung homogenates of P. brasiliensis-infected mice. BALB/c mice infected with P. brasiliensis were injected with emulsion PBS/MPLA (open circles) or PBS only (closed circles) on day 21 after infection. On day 30 after treatment, i.e., 50 days after infection, lung homogenates were used both to determine the fungal load by CFU assay and the copy number of the GP43 gene. DNA samples to quantify the GP43 gene were extracted from P. brasiliensis suspensions, which were obtained from lung homogenates free of host cells and cellular debris. Copy number was determined from a Cq standard curve, as described in Figure 1, with SYBR green (A and C) or TaqMan (B and D). A and B indicate a pre-test with five mouse and C and D experiments with 10 mice. The results represent the mean ± standard deviation values. Similar results to those of the C and D graphs were obtained in two independent experiments.

Thumbnail

Figure 1 Standard curves for qPCR - A) Correlation between the amount of genomic DNA from P. brasiliensis (log) and Cq. Four amounts of genomic DNA from P brasiliensis (ng) were submitted to qPCR using GP43 sequence-specific primers, which amplify 137 nt, and the DNA intercalating dye SYBR Green I. Cq (quantification cycle) consists of the number of cycles where there is the intersection of the cut line in the exponential phase of the amplification curve; B and C) Standard curves were constructed by plotting quantification cycle values (Cq) against an estimated DNA copy number to GP43 gene. Serial 10-fold dilutions of the plasmid pET28a-GP43 ranging from 2 ng to 2 fg (2.7 × 10⁸8. Buitrago MJ, Merino P, Puente S, Gomez-Lopez A, Arribi A, Zancope-Oliveira RM, et al. Utility of real-time PCR for the detection of Paracoccidioides brasiliensis DNA in the diagnosis of imported paracoccidioidomycosis. Med Mycol. 2009;47:879-82. to 2.7 × 10²2. Shikanai-Yasuda MA, Telles Filho FQ, Mendes RP, Colombo AL, Moretti ML. Consenso em paracoccidioidomicose. Rev Soc Bras Med Trop. 2006;39:297-310. copy number) were used as template in a qPCR run to construct the standard curves. The reactions were performed with the primers described above and either the dye SYBR Green I; B) or an allele-specific fluorogenic probe (TaqMan); C). All reactions were preformed in a CFX96 Touch Real-Time PCR Detection System. Error bars represent the standard deviation of duplicate PCR reactions.

Figure 2 The relationship between CFU per gram of lung and copy number of the GP43 gene obtained from the lung homogenates of P. brasiliensis-infected mice. BALB/c mice infected with P. brasiliensis were injected with emulsion PBS/MPLA (open circles) or PBS only (closed circles) on day 21 after infection. On day 30 after treatment, i.e., 50 days after infection, lung homogenates were used both to determine the fungal load by CFU assay and the copy number of the GP43 gene. DNA samples to quantify the GP43 gene were extracted from P. brasiliensis suspensions, which were obtained from lung homogenates free of host cells and cellular debris. Copy number was determined from a Cq standard curve, as described in Figure 1, with SYBR green (A and C) or TaqMan (B and D). A and B indicate a pre-test with five mouse and C and D experiments with 10 mice. The results represent the mean ± standard deviation values. Similar results to those of the C and D graphs were obtained in two independent experiments.

DNA (copy) = \frac{6.02 \times 10^{23} (copy / mol) \times DNA amount (g)}{DNA length (bp) \times 660 (g / mol / bp)}

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Quantitation of pulmonary fungal burden in <i>Paracoccidioides brasiliensis-infected</i> mice by real-time PCR

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[1] Correspondence to: Ademilson Panunto-Castelo Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Av. Bandeirantes, 3900, Monte Alegre, CEP 14040-901, Ribeirão Preto, SP, Brazil E-mail:apcastelo@usp.br