Detection of Streptococcus Pneumoniae and elimination of carryover contamination by multiple cross displacement amplication coupled with antarctic thermal sensitive uracil-DNA-glycosylase

Background: Streptococcus pneumoniae is an important clinical pathogenic bacterium, which is the primary cause of meningitis, septicemia and community-acquired pneumonia. The mortality rate of pneumococcal disease was high, especially in children under 5 years of age. Rapid and accurate detection of S. pneumoniae is critical. Methods: A ply gene-based multiple cross displacement amplication (MCDA), which amplies DNA under isothermal conditions (65 °C) for 40 min, was established for accurate and rapid detection of S. pneumoniae. Antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) was applied for eliminating carryover contamination. Lateral ow biosensor (LFB) was used to indicate the amplication results. Results: The ply-MCDA assay can detect as little as 10 fg of S. pneumoniae DNA, as well as 447 CFU/mL of spiked sputum samples. The sensitivity of ply-MCDA assay in clinical samples was 100 times that of PCR. The specicity of MCDA primer targeting the ply gene was validated using 15 S. pneumoniae and 25 non-S. pneumoniae, suggesting that ply gene-based MCDA assay was highly selective for S. pneumoniae. Moreover, the ply-MCDA coupled with AUDG can effectively eliminate carryover contamination, and then prevent false-positive results. Conclusion: The ply-MCDA assay coupled with AUDG was a simple, rapid and accurate method in the diagnosis of S. pneumoniae infection.


Background
Streptococcus pneumoniae is a gram-positive bacterium and regularly colonizes in the upper respiratory tract, which is the most common pathogen of human meningitis, septicemia, community-acquired pneumonia, otitis media and sinusitis [1,2]. According to the 2005 data of World Health Organization (WHO), S. pneumoniae infection causes 1.6 million deaths worldwide yearly, of which 700,000 to 1 million are children under 5 years of age, and mostly from developing countries [3]. Thus, rapid and accurate detection of S. pneumoniae is a primary task in preventing the spread of this pathogen and reducing the risk of pneumococcal disease.
The traditional detection technique for S. pneumoniae is bacterial culture-colonial morphologybiochemical examination, which is time-consuming, usually requiring three or more days. Rapid and accurate detection of S. pneumoniae is essential for clinical treatment. Polymerase chain reaction (PCR)based molecular biology techniques have been used for S. pneumoniae detection through primers speci c to ply gene [4][5][6]. Pneumolysin, which is encoded by the ply gene, is a virulence factor from S. pneumoniae and involved in the pathogenesis [7,8], representing the potential diagnostic target. However, the PCR reaction system is complex, complicated to perform, and requiring expensive thermal cycle apparatus and professional technicians [9]. Thus, PCR-based assay is not available in some backward clinical laboratories and "on-site" detection.
In recent years, a novel isothermal ampli cation method of DNA, multiple cross displacement ampli cation (MCDA), was devised by Wang et al. [10]. Compared with PCR, MCDA has superior performance, which ampli es DNA under isothermal temperature (60°C-69°C) with higher e ciency, speci city and only a simple thermostatic equipment is required. This method has been used for detecting Klebsiella pneumoniae [11], Vibrio parahaemolyticus [12], Listeria monocytogenes [13] and so on. The MCDA reaction system requires ten primers that specially recognize ten different regions of the target gene, and it can be completed within 40 min. Therefore, MCDA assay might be a valuable method for rapid and accurate detection of S. pneumoniae, especially when resource settings are limited. In addition, four detection methods of MCDA products, including colorimetric indicator, real-time turbidity, lateral ow biosensor (LFB) and gel electrophoresis have been adopted [14] .
In present study, MCDA assay was established for rapid and accurate detection of S. pneumoniae. Ply gene, S. pneumoniae-speci c gene that encodes pneumolysin, was used as the target gene. We evaluated the detection performance of ply-MCDA assay for S. pneumoniae. Furthermore, antarctic thermal sensitive uracil-DNA glycosylase (AUDG) was applied for the ply-MCDA reaction system to eliminate carryover contamination.

Primer design
Pneumolysin, a virulence factor, encoded by the ply gene, is a speci c target for S. Pneumoniae detection. Ten primers were designed targeting the ply gene of S. pneumoniae according to the primer design principle of MCDA [13]. Primer Primer 5.0 and PrimerExploer V4 were used. The pneumolysin (ply)encoding primer sets are listed in Table 3 and Figure 5. Blast analysis was performed to con rm the speci city of primer sets designed for S. pneumoniae. The 5'-ends of C1 was labeled with uorescein isothiocyanate (FITC). The 5'-ends of D1 was labeled with biotin. Primers synthesis was done in Aoke Dingsheng Biotechnology Co., Ltd. (Beijing, China).

Bacteria strains
In this study, 15 S. pneumoniae and 25 non-S. pneumoniae were used ( Table 1). The QIAamp DNA mini Kit (Hilden, Germany) was used for the extraction of genomic DNA from bacteria strains. Protocols were carried out according to the manufacturer's instructions. The S. pneumoniae ATCC49619 was used for performance con rmation, optimal temperature determination and sensitivity analysis. Serial dilutions of S. pneumoniae ATCC49619 DNA (10ng, 10pg, 1pg, 100fg, 10fg, 1fg and 0.1fg/μL) were used to analyze the sensitivity of ply-MCDA assay.
In this study, four methods including colorimetric indicator (Malachite Green, MG), turbidimeter (Loopamp Realtime Turbidimeter LA-320C), lateral ow biosensor (LFB) and 2% agarose gel electrophoresis were used to detect the ampli cation results. The above four methods were performed as previously described [14].
Optimal temperature of ply-MCDA assay The optimal temperature of S. pneumoniae ply-MCDA assay was measured at an isothermal temperature in the range of 62°C to 67°C at intervals of 1 °C. Distilled water was blank control. Staphylococcus aureus DNA and Salmonella typhi DNA were negative controls. The temperature that contributed to produce higher ampli cation products and occur turbidity earlier was considered for the optimal ampli cation temperature. This experiment repeated three times.
Sensitivity of ply-MCDA assay Sensitivity of ply-MCDA assay was determined using serial dilutions of S. pneumoniae ATCC49619 DNA. A total of 7 different concentrations of DNA templates (10ng, 10pg, 1pg, 100fg, 10fg, 1fg, 0.1fg/μL) were acquired. 1 μL of dilution was adding to the mixture system and then ampli ed at optimal temperature for 40min. Colorimetric indicator, real-time turbidity, LFB detection and gel electrophoresis were carried out to analyze the ply-MCDA products and then determine the detection limit of S. pneumoniae ply-MCDA assay. Distilled water was blank control. The sensitivity experiment repeated in triplicate.
AUDG enzyme eliminates carryover contamination. The S. pneumoniae ply-MCDA products without AUDG enzyme digestion were quanti ed and subsequently diluted in the range of 1×10−10 g/μL to 1×10−20 g/μL. These dilutions were used as simulated carryover contamination and served as templates in the S. pneumoniae ply-MCDA assay. To demonstrate that the simulated carryover contamination (UTP-incorporated ply-MCDA products) can contaminate the ply-MCDA reaction and con rm that AUDG enzyme can eliminate the carryover contamination, the sensitivity of ply-MCDA-AUDG and ply-MCDA were compared by using the above serial diluted S. pneumoniae ply-MCDA products (1 μL). The ultraviolet spectrophotometer (NanoDrop ND-1000, Calibre, China) was used for quanti cation.
Speci city of ply-MCDA assay To determine the speci city of S. pneumoniae ply-MCDA assay, genomic DNA from 40 bacteria strains including 15 S. pneumoniae and 25 non-S. pneumoniae were used for ply-MCDA assay ( Table 1). The ply-MCDA products were analyzed using colorimetric indicator and LFB. The speci city experiment repeated three times.
Clinical sensitivity of ply-MCDA assay The clinical sensitivity of S. pneumoniae ply-MCDA assay was evaluated by arti cially adding different amounts of colony forming units (CFU) of S. pneumoniae ATCC49619 to the sputum samples. The single colony of ATCC49619 was enriched and cultured. Then, the number of ATCC49619 CFUs was counted and the culture suspension was added to S. pneumoniae negative sputum samples. The concentration of ATCC49619 in the sputum samples was adjusted to 4.47×100, 4.47×101, 4.47×102, 4.47×103, 4.47×104, 4.47×105, 4.47×106 CFU/mL. 100 μL of each spiked sputum sample was taken for genomic DNA extraction and then eluted in 10 μL of elution buffer (Qiagen, Germany). Next, 1 μL of DNA template was used for ply-MCDA assay and PCR method was also adopted. The experiment was performed three times independently.

Results
Con rmation of ply-MCDA products.
To con rm the feasibility of the ply gene-targeted MCDA assay in detecting S. pneumoniae, ply-MCDA assay with and without target DNA were conducted at 65 º C for 40 min. The ply-MCDA products of S. pneumoniae ATCC49619 was visually detected as lake green color in the tube (Fig.1A), two red lines (control line and test line) on the LFB (Fig.1B) and ladder-like bands on the gel (Fig.1C), which indicated the ampli cation with target sequence. While the ply-MCDA products of negative controls and blank control were present as colorless (Fig.1A), one red line (control line) (Fig.1B) and non-ladder bands (Fig.1C). Our results suggested that S. pneumoniae-speci c primers targeting the ply gene were suitable for MCDA assay.
Optimal temperature of ply-MCDA assay.
To measure the optimal temperature of the ply-MCDA assay, the DNA of S. pneumoniae ATCC49619 (1 pg/reaction) was ampli ed at intervals of 1 °C in the range of 62°C to 67°C, respectively. The real-time turbidity of different temperatures was monitored. From the Kinetic curves as shown in Figure 2, we found that the optimal ampli cation temperature was 65°C because the reaction turbidity occurred earlier and the amount of ply-MCDA products were higher than other temperatures (Fig. 2D). 65°C was used in subsequent studies.
To identify the detection limit, serial dilutions of S. pneumoniae ATCC49619 DNA (10ng, 10pg, 1pg, 100fg, 10fg, 1fg, 0.1fg/μL) were subjected to be ampli ed at 65°C. Our results indicated that the detection limit of S. pneumoniae ply-MCDA assay was 10fg (Fig.3). The positive amplicons were represented in the color indicator (Fig.3A), real-time turbidity (Fig.3B), LFB (Fig.3C) and gel electrophoresis (Fig.3D) as lake green color, turbidity curve, two red lines and ladder-like bands, respectively. Moreover, the results of the above four methods were completely consistent.
To determine whether the carryover contamination from the UTP-incorporated ply-MCDA products would contaminate the ply-MCDA reaction, the sensitivity of ply-MCDA-AUDG and ply-MCDA were evaluated by using serial dilutions of ply-MCDA products (between 1×10−10 g/μL and 1×10−20 g/μL). Our results showed that ply-MCDA-AUDG could detect simulated carryover contamination of 1×10−14 g/μL or more (Fig.4). However, ply-MCDA without AUDG enzyme could detect simulated carryover contamination of 1×10−19 g/μL (Fig.4). These results indicated that false positive results would occur when the concentration of carryover contamination reach 1×10−19 g/μL. In addition, the results demonstrated that the AUDG enzyme is able to eliminate the carryover contamination. Therefore, the possibility of false positive results can be signi cantly reduced by adding AUDG enzyme to the ply-MCDA system.
Speci city of ply-MCDA assay.
To identify the speci city of ply-MCDA assay with AUDG digestion in detecting S. pneumoniae, DNA from 15 S. pneumoniae and 25 non-S. pneumoniae were tested. The 15 strains of S. pneumoniae showed positive results, while other Streptococcus species and non-Streptococcus strains showed negative results ( Table 1). The results suggested that ply-MCDA-AUDG assay was highly speci c for S. pneumoniae.
Clinical sensitivity of ply-MCDA assay.
To determine the sensitivity of ply-MCDA assay with AUDG digestion in clinical samples, sputum samples arti cially contaminated with serial dilutions of S. pneumoniae ATCC49619 were tested. Positive ampli cation occurred when the amount of S. pneumoniae ATCC49619 in the sputum samples reach 447 CFU/ml (equivalent to 4.47 CFU per reaction) ( Table 2). Moreover, the sensitivity of ply-MCDA-AUDG assay was 100 times that of PCR, the detection limit of PCR was 44700 CFU/ml (equivalent to 447 CFU per reaction) ( Table 2). Discussion S. pneumoniae, an important human pathogenic bacterium, causes high morbidity and mortality. Statistics show that in China, the cases of pneumococcal infection account for 12% of the global cases, which is also one of the countries with the highest deaths caused by pneumococcal infection in children under 5 years old [15]. Because of the severity of S. pneumoniae infection, simple and accurate methods are needed to detect S. pneumoniae and timely guide clinical treatment. Mitsuko et al. has reported a rapid method, loop-mediated isothermal ampli cation (LAMP), applying for detection of S. pneumoniae infection, which is highly selective due to its recognition of six different regions of the target gene by four primers [16,17]. Studies have reported that increasing the primers of isothermal ampli cation could improve the sensitivity [18]. This study developed a novel method, MCDA in combination with AUDG, to speci cally detect S. pneumoniae and prevent false positive results.
Within the present study, we designed ve pairs of primers targeting ten distinct regions of the ply gene. Ply gene, which encodes pneumolysin, a well-characterized virulence factor of S. pneumoniae and participating in the pathogenesis, has been used as the speci c target gene of S. pneumoniae [7,8] . Our results demonstrated that the primer sets are highly speci c for S. pneumoniae. All of the strains of S. pneumoniae were positively ampli ed, while the other Streptococcus species and non-Streptococcus strains were not ampli ed (Table 1). Therefore, the speci city of ply-MCDA assay in the diagnosis of S. pneumoniae infection was extremely high, especially combining with clinical manifestations.
In addition, we determined the sensitivity of ply-MCDA assay by testing serial dilutions of S. pneumoniae ATCC49619 DNA. The detection limit of ply-MCDA assay was 10fg (Fig.3). It has been reported that LAMP can detect S. pneumoniae as few as 25 fg [19] or 20 fg [20]. We also assessed the clinical sensitivity of this assay by using spiked sputum samples. The ply-MCDA assay can detect S. pneumoniae as low as 447 CFU/ml (equivalent to 4.47 CFU per reaction), which is similar to that of LAMP [19]. Thus, ply-MCDA assay was a sensitive method for S. pneumoniae detection.
However, owing to the high sensitivity of ply-MCDA assay, false positive results caused by carryover contamination became a barrier to rapid and accurate detection of S. pneumoniae. Moreover, aerosols that containing high concentrations of products cannot be avoided because the opening indicating operations of ply-MCDA products (such as LFB). The results of this study suggested that trace amounts of carryover contamination (1×10−19 g/μL) can lead to false positive results. Thus, AUDG enzyme digestion was performed to prevent the false positive results caused by carryover contamination, which speci cally cleave uracil bases and permit nature templates to be ampli ed normally [11,19], and which can eliminate up to 1×10−15 g/μL of simulated contaminants. These results suggested that ply-MCDA-AUDG assay can effectively eliminate carryover contamination and then reduce false positive results.
About results analysis, colorimetric indicator, real-time turbidity, LFB and gel electrophoresis were used. Our results showed that these four methods were highly consistent. However, comparing with conventional analysis methods, LFB was probably the optimal monitoring method. In the MCDA system, one primer was labeled with FITC and one primer was labeled with biotin. Subsequently, positive amplicons are simultaneously labeled with FITC and biotin. In LFB, FITC and biotin labeled products combined with dye streptavidin coated on the control line and anti-FITC antibodies immobilized on the test line, respectively. The results are displayed as two red lines within 2 minutes. The advantages of LFB included simple, rapid, cost-effective, objective, and no equipment requirement. Thus, LFB is an effective method for analysis of MCDA products.
In summary, we established a simple, rapid and accurate ply-MCDA-LFB method in combination with AUDG for the diagnosis of S. pneumoniae and eliminating carryover contamination. LFB provided a potential tool for rapid and accurate detection of S. pneumoniae. The sensitivity, speci city and clinical sensitivity of ply-MCDA assay in the diagnosis of S. pneumoniae were successfully evaluated using cultured strains and sputum samples. Therefore, the ply-MCDA-LFB method coupled with AUDG is potentially a valuable tool for the detection of S. pneumoniae.   Optimal temperature of ply-MCDA assay. The DNA templates of S. pneumoniae ATCC49619 (1pg/reaction) were ampli ed at 62°C to 67°C (1°C intervals), respectively. The real-time turbidities were monitored at 650 nm (A-F). Staphylococcus aureus DNA and Salmonella typhi DNA were negative controls, and distilled water were blank control. Turbidity>0.1 was de ned as positive.  AUDG enzyme eliminates carryover contamination. The sensitivity of ply-MCDA assay with AUDG (AUDG+) and without AUDG (AUDG-) were evaluated using 10-fold serial diluted simulated carryover contamination (UTP-incorporated ply-MCDA products, concentrations between 1×10−10 and 1×10−20 g/ μL). The products were analyzed by colorimetric indicator (A) and LFB (B).