SYBR Green qPCR Technique for the Detection of Trypanosoma cruzi in Açaí Pulp

Abstract

Chagas disease, which is found widely in Latin America and has a great impact on public health, is caused by the parasite Trypanosoma cruzi. It is a neglected parasitic disease that urgently requires rapid diagnostic methods. The objective of this study was to develop a SYBR Green real-time quantitative polymerase chain reaction (qPCR) technique for the direct identification and quantification of T. cruzi from experimentally contaminated açai fruit samples. We used discrete typing units, TcI, containing 3.5 × 10⁴ cells/mL, to infect the pulp of the açai fruit. This was followed by DNA extraction using a standardized procedure. The DNA samples were quantified and amplified at specific time and temperature intervals. The specificity of the oligoinitiators used in the qPCR assays was estimated by calculating the primer dissociation curve (melting curve) along with a detection threshold using different concentrations of DNA. The method used here demonstrated good efficiency and precision for the detection and quantification of T. cruzi DNA, with a detection limit of 2.65 × 10⁻¹⁴ g/μL DNA. The qPCR technique presented here could serve as an important tool for the diagnosis of T. cruzi parasites in açai.

Introduction

The protozoan Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas disease (CD), a systemic disease that is often asymptomatic (WHO, 2010). In northern Brazil, oral transmission of T. cruzi is frequent, particularly in the Pará and Amapá states, where CD outbreaks are associated with consumption of contaminated açai (Nóbrega et al., 2009; Filigheddu et al., 2017; Shikanai-Yasuda, et al., 2018; Brazil, 2019) via feces of the triatomine vector.

The states in the northern regions of Brazil (especially Pará and Amapá) are major producers of açai, accounting for 98% of the national production, with most of it destined for export (IBGE, 2017).

Contamination of Brazilian acai represents a risk to public health worldwide, since this food is marketed in Brazil in abundance, and its consumption by tourists may increase the risk of T. cruzi transmission to these people, which may lead to an increased rate of infected individuals in nonendemic countries. This may also lead to spread of T. cruzi through other routes of transmission such as via blood transfusion and congenital route (Ferreira et al., 2014).

In addition, most of the acai berry consumed in the world is of Brazilian origin and, although this product it can be pasteurized, this procedure is generally not performed due to organoleptic changes of the product (Barbosa et al., 2016). Thus, control of this raw material in Brazil would help ensure product safety and market supremacy.

Due to the importance of this disease and difficulties in identifying T. cruzi, previous studies have tried to optimize polymerase chain reaction (PCR) assays for parasite detection in both human samples (Seiringer et al., 2017; Moreira and Ramirez, 2019) and contaminated açai (Godoi et al., 2017; Ferreira et al., 2018). However, studies that focus on detection of T. cruzi directly from açai samples are scarce, and they have detection thresholds that can be improved.

Among the PCR techniques available, SYBR Green^® qPCR stands out as a quantitative and sensitive method. It also offers the advantages of being cost-effective, easy to execute, and can provide real-time monitoring of results (Tajadini et al., 2014). This technique also allows for identification and quantification of T. cruzi DNA in contaminated açai samples. Although studies have demonstrated the efficiency of this methodology (Godoi et al., 2017), the detection limit of T. cruzi DNA by SYBR Green qPCR can be improved.

Therefore, this study aimed at validating a real-time qPCR assay using SYBR Green to detect T. cruzi in experimentally contaminated açai samples, and it can provide a viable quality control option for commercial samples.

Materials and Methods

T. cruzi cultivation

For this study, a strain of T. cruzi discrete typing units TcI, obtained from the protozoology collection of the Oswaldo Cruz Institute Foundation in Rio de Janeiro, Brazil was used. The parasite was grown in liver infusion tryptose medium, and it was kept in a greenhouse at 28°C, ad eternum.

The parasite cells were collected from cell cultures, suspended in phosphate-buffered saline (PBS) at pH 7.2, and counted in the Neubauer chamber. A stock solution containing 3.0 × 10⁴ cells/mL was used to prepare the experimentally contaminated açai.

DNA extraction

To validate the qPCR proposal, we optimized T. cruzi DNA extraction from contaminated açai. The Illustra™ Tissue and Cells genomicPrep Mini Spin Kit (GE Healthcare, Buckinghamshire, UK) were used according to the manufacturer's protocol, with some modifications; these included addition of a washing step where 100 mL PBS was added to a 200-mL sample and centrifuged at 14,000 g for 2 min. The lysis, precipitation, and purification steps were performed according to the manufacturer's recommendations. DNA from an acai sample that was not contaminated with T. cruzi was extracted by using the same protocol, and it served as negative control. Extracted DNA was suspended in 50 μL of hydration solution at 37°C for 5 min, and it was stored at 4°C until further use.

The extracted DNA was quantified by using a Nanodrop Lite^® spectrophotometer at 260 nm (A260), 280 nm (A280), and 230 nm (A230) to determine the quality and concentration of the genetic material.

The integrity of the DNA was evaluated by using 1% agarose gel electrophoresis, which was run in Tris Borate Ethylenediamine tetraacetic acid buffer; 1 μL of 6 × Gelred™ (Biotium^©, Fremont) was mixed with 3 μL of sample. Results were analyzed by using ultraviolet light photo documentation equipment (Gel Documentation System, Gel Doc Xr+™; Bio-Rad).

SYBR Green qPCR technique

For SYBR Green qPCR, two primers previously described by Ochs et al. (1996), TCZ3 (5′-TGCTGCASTCGGCTGATCGTTTTCGA-3′) and TCZ4 (5′-CARGSTTGTTTGGTGTCCAGTGGTTGTG-3′), were used to amplify 149 base pairs (bp) sequence; primers were eluted in TE buffer (pH 8.0) at 100 pmol/μL concentration, according to the manufacturer's recommendations (Ludwig biotec^©, Rio Grande do Sul, Brazil). Each reaction was prepared by using a mix of reagents containing 10 μL of the previously extracted T. cruzi DNA (∼1.336 × 10⁻⁷ g/μL), 12 μL of qPCR-SYBR-Green mix/ROX (Ludwig biotec, Rio Grandre do Sul, Brazil), and 5 pmol of each primer to obtain a final volume of 24 μL.

DNA extracted directly from T. cruzi was used as a positive control. Sterile ultrapure water was used as the negative control.

Assays were performed on 96-well semi-contoured polyethylene plates (Corning^® Nova York) by using a StepOnePlus™ Real-Time PCR (Applied Biosystems, Foster City, CA) system under the following conditions: pre-incubation at 48°C for 5 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 57°C for 1 min.

Melting curve analysis was done immediately after the final amplification step by heating the samples at 95°C for 15 s, cooling to 60°C for 15 s, heating to 95°C for 15 s, and finally at 60°C for 15 s. Melting curves were recorded by plotting fluorescence signal intensity versus temperature. DNA amplification, data acquisition, and analysis were all completed by using StepOnePlus v2.3.

Analysis of standard curve

A standard curve was built with fivefold serial dilutions of T. cruzi DNA from 1.36 × 10⁻⁷ g/μL to 1.36 × 10⁻¹² g/μL, performed in triplicate. The standard curve shows cycle threshold (Ct) values on the y-axis and logarithmic genomic equivalent (GE) concentration on the x-axis. The standard curve was calculated by using the DNA Copy Number tool and Dilution Calculator, available at www.thermofisher.com.

The linear regression formula generated could be used to calculate the concentrations of unknown samples, using the regression formula (Y = ax + b). In addition, reaction efficiency was calculated from the formula E = (10^[−1/slope]−1) by using the standard curve data.

Evaluation of technique sensitivity

Once the methodology was standardized, its technical detection limits were determined. For this, Euterpe oleracea fruits were harvested in a rural farm in the municipality of Castanhal, state of Pará, Brazil. Fruits were blanched (heating at 80°C for 10 s, followed by cooling to 7°C) and processed by specific equipment into 9-mL pulp samples.

Parasite cells were cultured, and stock solution containing 3.0 × 10⁴ cells/mL were diluted to 10⁻¹⁰ cells/mL; 1 mL of the diluted suspension was added to 9-mL pre-bleached açai pulp. DNA samples were subjected to the aforementioned qPCR assay. All tests were performed in quadruplicate.

Results

The results of the proposed qPCR showed that the technique is efficient for the detection of T. cruzi DNA when an annealing temperature of 57°C was used. The qPCR efficiency tested was found to be 103.98%. Samples were considered to be positive when the Ct value was less than 35, and when amplification was detected for all replicates. The melting curve analysis showed a single peak, and the melting temperature (Tm) was 80°C.

The optimized protocol was adapted for DNA extraction directly from the contaminated açai berry to ensure adequate quality. Based on the results, the lower detection limit was 2.6 × 10⁻¹⁴ g/μL DNA, as shown in Table 1, which corresponds to 1.87 × 10⁷ GE. This demonstrated the ability of the assay to detect DNA at low concentrations, which suggested that a few specimens are needed for sample detection.

Table 1.

Trypanosoma Cruzi Detection Limit Using Quantitative Polymerase Chain Reaction in Açai Samples

Dilution	[DNA] (g/μL)	Genomic equivalence	Ct	qPCR result^a
10⁰	2.65 × 10⁻⁷	1.65 × 10¹²	31	+
10⁻¹	2.65 × 10⁻⁸	1.65 × 10¹¹	33	+
10⁻²	2.65 × 10⁻⁹	1.65 × 10¹⁰	33	+
10⁻³	2.65 × 10⁻¹⁰	1.65 × 10⁹	34	+
10⁻⁴	2.65 × 10⁻¹¹	1.65 × 10⁸	34	+
10⁻⁵	2.65 × 10⁻¹²	1.65 × 10⁷	34	+
10⁻⁶	2.65 × 10⁻¹³	1.65 × 10⁶	35	+
10⁻⁷	2.65 × 10⁻¹⁴	1.65 × 10⁵	35	+
10⁻⁸	2.65 × 10⁻¹⁵	1.65 × 10⁴	38	−
10⁻⁹	2.65 × 10⁻¹⁶	1.65 × 10³	38	−
10⁻¹⁰	2.65 × 10⁻¹⁷	1.65 × 10²	38	−

+ positive; − without amplification; [·] DNA concentration.

The result was considered positive when all replicates tested were amplified for each dilution.

Ct, cycle threshold; qPCR, quantitative polymerase chain reaction.

The cost of sample analysis including DNA extraction reagents was approximately US$ 5.95 for one sample, and the cost of reagents used in SYBR Green was US$ 0.34 per sample.

Discussion

Among the PCR techniques, real-time qPCR stands out for its greater sensitivity, specificity, and speed. Further, it can quantify DNA sequences and identify low concentrations of genetic material. Therefore, the development of a qPCR protocol for the diagnosis of the T. cruzi is crucial to provide a tool that is able to assess the effectiveness of treatment and diagnosis in CD patients, quantify the parasitic burden in infected tissues (Martínez et al., 2011; Duffy et al., 2013; Moreira et al., 2013; Rendell et al., 2015), and identify parasitic agents in contaminated food.

In the cases of oral infection via consumption of acai juice, contamination may occur by fecal deposition of T. cruzi in the fruit or by maceration of Triatomines infected by the agent during fruit pulp processing (Xavier et al., 2014). Thus, standardization of qPCR from samples contaminated experimentally with infecting parasites may be useful for detection of contaminated acai by both vector insects and their feces. Future work on açai samples contaminated with infected Triatomine should also be performed.

To obtain T. cruzi DNA directly from açai samples, several authors previously proposed qPCR methods (Mattos, et al., 2017; Ferreira et al., 2018) by using TaqMan^® dye.

Studies performed by Godoi et al. (2017) used the SYBR Green method when optimizing a qPCR assay for detection of T. cruzi in açai. However, although their results are significant, they demonstrated a high detection limit by using the proposed methodology. The authors of this study were able to identify only 0.44 parasite molecules, whereas this study was almost 150 times more sensitive. The authors of this study were only able to identify the pathogen molecules at a parasitic load of 0.44. The difference between these two results can be explained by the fact that Godoi et al. (2017) extracted DNA via the phenol/chloroform method, which, according to Silva et al. (2015), may reduce the efficiency of PCR. Another factor that hinders the development of an appropriate qPCR technique is the choice of primers. Godoi et al. (2017) tested six different primer pairs, but they were unable to obtain better results than those presented here. Their best result was 0.1 pg/μL DNA. Study results showed that these primers exhibited a minimal amplification limit of 2.65 × 10⁻¹⁴ g/μL DNA.

Thus, our work is the first to demonstrate a qPCR technique with high efficiency and low cost using SYBR Green as the dye. This method can be considered a viable alternative to detecting T. cruzi directly from the pulp of açai. The difficulty in developing conventional or real-time PCR protocols for açai pulp is largely due to the presence of inhibitors in the fruit that hinders the process of DNA extraction. Ferreira et al. (2016) also report on this fact.

The need to validate this methodology lies in the fact that it needs to have the diagnostic sensibility to detect parasitic contamination in food. There are still difficulties associated with this technique that need to be overcome. The main obstacles, according to Passos et al. (2012), are related to the dark coloration of the fruit, the high levels of organic matter, especially vegetable fibers, and the physicochemical characteristics of the pulp of the fruit.

According to Mattos et al. (2017), the composition of the foods being analyzed may interfere with the process of obtaining genetic material of an adequate purity. Since many contaminants and inhibitors of reactions may be present in food matrices, therefore, to optimize the technique, it is necessary to develop or adapt more efficient extraction protocols to obtain more DNA. In this study, we propose adaptations to a commercial kit that enables extraction of high-quality DNA suitable for qPCR assays. This is possible, for example, by eliminating a large part of the organic contaminants of proteins found in food.

Ferreira et al. (2016) reported on the challenges in obtaining T. cruzi DNA in a food matrix as complex as that of açai. The identification of the parasite's genetic material in the food product provides evidence that good practices have been applied during the manufacturing of the product. Thus, our DNA extraction results demonstrate a viable alternative whenever genetic material is needed for the study of a target fruit.

In our work, the melting curve analysis showed a single peak at the Tm, which suggested that the SYBR Green qPCR assay had good sensibility. Several other authors have also previously demonstrated the effectiveness of qPCR in the detection of T. cruzi directly from açaí samples.

Validation of qPCR assays for detection of T. cruzi, as presented here, is of paramount importance, since it allows us to optimize diagnosis of CD, which can push for policies that enhance disease prevention, as stated by Schijman (2018). In the same context, Teston et al. (2017) previously stressed the importance of CD cases via oral infection, which generate high levels of parasitemia and result in higher tissue parasitic load. These authors indicated that qPCR is the most sensitive method for detection of T. cruzi, which reinforces the relevance of our study. Thus, the qPCR amplification technique proposed in this study is of great importance, especially in Brazil, which has the greatest exports of açai consumed around the world (IBGE, 2017).

qPCR assays using SYBR Green can be performed without compromising the efficiency or specificity of the reaction, resulting in a methodology that is one of the most cost-effective and viable techniques among those currently available in scientific literature. The methodology shown in this study will contribute to the fields of both science and medicine.

Conclusions

We conclude that the proposed qPCR was efficient and has a low detection limit, demonstrating that our protocol using SYBR Green may be an alternative for quality control of commercial samples.

Footnotes

Acknowledgments

The authors would also like to thank the Protozoology Collection of the Oswaldo Cruz Institute Foundation for provision of the positive control used in their experiments.

Disclosure Statement

No competing financial interests exist.

Funding Information

The authors are grateful for Coordination for the Improvement of Higher Education Personnel (CAPES: scholarship), National Council for Scientific and Technological Development (CNPq: 448720/2014), and Pro-Rectory of Research and Postgraduate of the Federal University of Pará (PROPESP: 23073.008095/2019-70) for their financial support.

References

Barbosa

, Pereira

, Dias

, Schmidt

, Alves

, Guaraldo

AMA

, Passos

LAC

. Virulence of Trypanosoma cruzi in açai (Euterpe oleraceae Martius) pulp following mild heat treatment. J Food Prot, 2016; 79:10.

Brazil. Acute Chagas' disease and spatial distribution of triatomines of 18 epidemiological importance (language portuguese), Brasil 2012 a 2016. Bol Epidemiol, 2019; 50:2.

Duffy

, Cura

, Ramirez

, et al. Analytical performance of a multiplex real-time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples. PLoS Negl Trop Dis, 2013; 7:2000e.

Ferreira

RTB

, Branquinho

, Leite

. Oral transmission of Chagas disease by consumption of acai: A challenge for health surveillance. Vig Sanit Debate, 2014; 2:4–11.

Ferreira

RTB

, Melandre

, Cabral

, Branquinho

, Cardarelli-Leite

. Detection and genotyping of Trypanosoma cruzi from açai products commercialized in Rio de Janeiro and Pará, Brazil. Parasit Vectors, 2018; 11:233.

Ferreira

RTB

, Melandre

, Cabral

, Branquinho

, Cardarelli-Leite

. Extraction of Trypanosoma cruzi DNA from food: A contribution to the elucidation of acute Chagas disease outbreaks. Rev Soc Bras Med Trop, 2016; 49:190–195.

Filigheddu

, Górgolasb

, Ramosd

. Orally-transmitted Chagas disease. Med Clin 17 (Barc), 2017; 148:125–131.

Godoi

PSA

, Piechnik

, Oliveira

, Sfeird

, Souza

, Rogezc

, Soccolb

. qPCR for the detection of foodborne Trypanosoma cruzi . Parasitol Int, 2017; 66:563–566.

IBGE—Instituto Brasileiro de Geografia e Estatística. Censo Agropecuário. 2017. Available at: https://sidra.ibge.gov.br/tabela/6617#resultado, accessed May 30, 2018 .

10.

Martínez

, Quilez

, Sánchez

, Roura

, Francino

, Altet

. Canine leishmaniasis: The key points for qPCR result interpretation. Parasit Vectors, 2011; 4:2–5.

11.

Mattos

, Meira-Strejevitch

, Marciano

MAM

, Faccini

, Lourenço

, Pereira-Chioccola

. Molecular detection of Trypanosoma cruzi in acai pulp and sugarcane juice. Acta Trop, 2017; 176:311–315.

12.

Moreira

, Ramirez

. Genotyping of Trypanosoma cruzi from clinical samples by multilocus conventional PCR. Methods Mol Biol, 2019; 1955:227–238.

13.

Moreira

, Ramírez

, Velázquez

, et al. Towards the establishment of a consensus real-time qPCR to monitor Trypanosoma cruzi parasitemia in patients with chronic Chagas disease cardiomyopathy: A substudy from the BENEFIT trial. Acta Trop, 2013; 125:23–31.

14.

Nóbrega

, Garcia

, Tatto

, Obara

, Costa

, Sobel

, et al. Oral transmission of Chagas disease by consumption of açai palm fruit, Brazil. Emerg Infect Dis, 2009; 15:653–655.

15.

Ochs

, Moser

, Smith

, Kirchhoff

. Postmortem diagnosis of autochthonous acute chagasic myocarditis by polymerase chain reaction amplification of a species-specific DNA sequence of Trypanosoma cruzi . Am J Trop Med Hyg, 1996; 54:526–529.

16.

Passos

LAC

, Guaraldo

AMA

, Barbosa

, Dias

, Pereira

, Schmidt

, et al. Trypanosoma cruzi survival and infectivity in acai pulp: In vitro and in vivo study. (Language portuguese). Epidemiol Serv Saúde, 2012; 21:223–232.

17.

Rendell

, Gilman

, Valencia

, et al. Trypanosoma cruzi-infected pregnant women without vector exposure have higher parasitemia levels: Implications for congenital transmission risk. PLoS One, 2015; 10:e0119527.

18.

Schijman

. Molecular diagnosis of Trypanosoma cruzi . Acta Trop, 2018; 184:59–66.

19.

Seiringer

, Pritsch

, Flores-Chavez

, et al. Comparison of four PCR methods for efficient detection of Trypanosoma cruzi in routine diagnostics. Diagn Microbiol Infect Dis, 2017; 88:225–232.

20.

Shikanai-Yasuda

, Carvalho

. Oral transmission of Chagas disease. Clin Infect Dis, 2018; 54:845–852.

21.

Silva

, Sales

, Santos Neto

, Silva

, Lara

APSS

, Lima

SCG

, et al. Detection of fraud in commercial samples of buffalo cheese by adding bovine milk using the Multiplex Polymerase Chain Reaction (PCR) technique. (Language portuguese). Rev Inst Adolfo Lutz, 2015; 74:1.

22.

Tajadini

, Panjehpour

, Javanmard

. Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes. Adv Biomed Res [serial on-line], 2014; 3:85.

23.

Teston

AMP

, Abreu

, Abegg

, Gomes

, Toledo

MJO

. Outcome of oral infection in mice inoculated with Trypanosoma cruzi IV of the Western Brazilian Amazon. Parasitol Int, 2017; 66:563–566.

24.

WHO-World Health Organization. Working to Overcome the Global Impact of Neglected Tropical Diseases: First WHO Report on Neglected Tropical Diseases. Geneva: World Health Organization, 2010.

25.

Xavier

SCDC

, Roque

ALR

, Bilac

, de Araújo

VAL

, Neto

SFDC

, et al. Distantiae transmission of Trypanosoma cruzi: A new epidemiological feature of acute Chagas disease in Brazil. PLoS Negl Trop Dis, 2014; 8:e2878.