Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61–65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.


Confirmation and detection of MCDA products.
To confirm the correct amplification of MCDA, amplification systems were performed in the presence or absence of genomic DNA templates at 63°C. Two determination methods, including colorimetric indicators (such as FD reagent) and agarose gel electrophoresis, were selected for monitoring the MCDA reaction 7 .
The FD reagent can be added directly during the MCDA reaction mixture preparation, and indicates the result of the MCDA reaction by the color change. Thus, the positive results can be detected from the change of color by the naked eye. In the MCDA tubes, the color change of positive reactions from light gray to green was directly observed by naked eye, while the negative control tube remained light gray (Fig. 3A).
The final products of the MCDA reaction are mixtures of various short segments of DNAs ( Fig. 1  structures 3, 4, 5 and 7) and stem-loop DNAs with different stem lengths formed by annealing between alternatively inverted repeats of the target DNA in the same sequence ( Fig. 1 structures 6 , 8, 9, 10 and 11). Therefore, electrophoretic analysis of the MCDA amplified products, the characteristic ladder-liker pattern bands on agarose gel electrophoresis should be yielded in positive amplification but not in the negative control. As expected, the positive results were observed many bands of different sizes in a ladder-like pattern on ethidium bromide-stained 2.5% agarose gel electrophoresis (Fig. 3B). These results suggested that the novel MCDA technique could correctly amplify DNA under isothermal conditions. Validation of the reliability of MCDA by sequencing. To further confirm the reliability and specificity of MCDA assay, a MCDA-PCR-sequencing strategy was developed to analyze the MCDA products. The sequences of PCR amplicons, which amplified from MCDA products, were 98% match with the expected sequences (Fig. 4). The sequencing data showed that the correct amplification of MCDA was further validated.
The optimal temperature of MCDA assay. For an isothermal amplification assay, the key is the establishment of a dynamic reaction environment at a constant temperature that balances binding of the primer to the template strand with elongation activity of the polymerase. Herein, the MCDA reactions were tested at different temperatures (60-67 °C) with the appropriate primers and the reference strain EGD-e was selected as positive control to determine the optimal temperature at the level of 250 pg genomic DNA per reaction. The results were analyzed by real-time measurement of turbidity and a typical kinetics graph was shown in Fig. 5. The positive products were also visualized as a ladder-like banding pattern on agarose gel electrophoresis (Fig. 6). The reaction temperatures of 61-65 °C were recommended as the standard temperatures for the MCDA assay and the temperature of 63 °C was selected for the rest of MCDA amplification conducted in this study.   Fig. 7A, the MCDA reactions were monitored by real-time turbidity detection; the decreasing amounts of DNA were listed from left to right and the LoD of MCDA assay was 62.5 fg DNA per reaction. The positive reactions by FD reagent were visualized as the change of color from light gray to green, and the positive results by 2.5% agarose gel electrophoresis were seen as a ladder-like pattern (Fig. 7B,C). The LoD of the two monitoring methods for MCDA reaction was 62.5 fg DNA per reaction, which was identical with turbidity detection. Moreover, the MCDA reactions only require 40-min incubation periods at LoD levels of genomic DNA (Fig. 7A). The LoD of LAMP and CPA reactions on L. monocytogenes was 1 pg DNA/tube and 2 pg DNA/tube, respectively, whereas the LoD of MCDA assay was 62.5 fg DNA/tube (Fig. 8, Table 2). These results showed that the MCDA approach was 16-fold and 32-fold more sensitive than the LAMP and CPA assays for detecting L. monocytogenes genomic DNA, respectively. Moreover, the LAMP and CPA reactions required 52-and 63-min incubation periods at LoD levels of template DNA, respectively ( Table 2).

Specificity of the MCDA assay.
To evaluate the specificity of the MCDA approach, the L. monocytogenes MCDA assay was carried out under the standard conditions described above with the genomic DNA extracted from the 40 non-L. monocytogenes and 50-L. monocytogenes strains ( Table 3). The positive results by FD reagent were directly seen as the color change in all L. monocytogenes strains, but not in non-L. monocytogenes strains (Fig. 9A). As shown in Fig. 9B, the typical ladder-like pattern on 2.5% agarose gel electrophoresis was produced in all L. monocytogenes strains, whereas the non-L. monocytogenes strains did not show the characteristic pattern. These results indicated that the L. monocytogenes MCDA assay was highly specific to the pathogen identification.
Practical application of MCDA to L. monocytogenes detection in pork samples. For the purpose of demonstrating the applicability of the MCDA assay as a nucleic acid analysis tool, the L. monocytogenes-MCDA method was applied to detect the foodborne pathogen in pork samples. Fifty-five pork samples were analyzed using PCR, MCDA and traditional culture-biotechnical assays. The results were summarized in Table 4. Of a total of 55 pork samples, 14 (25.5%) were L. monocytogenes positive by the novel MCDA assay which was in accordance with the results by culture-biotechnical method, and 8 (14.5%) for the conventional PCR method. These results suggested that the proposed methodology was a good alternative to conventional culture-biotechnical technology and could provide a potential for nucleic acid analysis.
In order to demonstrate the correct amplification of S-MCDA, two monitoring methods, including FD reagent and agarose gel electrophoresis, were applied to determine the S-MCDA reactions. A positive color (green) was visualized in the presence of L. monocytogenes genomic DNA, a negative color (light gray) for the control tube ( Fig. 10A,C). The positive DNA products were observed as a ladder-like banding pattern on 2.5% agarose gel electrophoresis (Fig. 10B,D).
The sensitivity of S 1 -MCDA and S 2 -MCDA reactions was also assessed by analyzing the products generated from the serial dilutions (2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 125 fg, 62.5 fg and 31.25 fg per microliter) of the L. monocytogenes genomic DNA, and three replicates of each dilution were tested. The LoD of S 1 -MCDA and S 2 -MCDA was 125 fg DNA/reaction (Fig. 11). The positive results of S 1 -MCDA and S 2 -MCDA were produced in as short as 25 minutes and the two S-MCDA reactions required 50-min incubation periods at the LoD levels of DNA (Fig. 11A,B). The specificity of S 1 -MCDA and S 2 -MCDA reactions was determined using the genomic DNA extracted from 50 L. monocytogenes and 40 non-L. monocytogenes strains. The DNA of all L. monocytogenes was tested positive with the two sets of primers, whereas no amplification of the non-L. monocytogenes genomic DNA was observed (data not shown).

Discussion
The adoption of nucleic acid amplification in vitro has resulted in powerful technologies that underpinned biological research and diagnostics. Because of the requirement for complicated apparatus, special reagents and multiple enzymes, these techniques were used as special tools available only to particular fields, facilities and laboratories 8 . However, these technologies are constantly evolving and the design of alternative methodology for simple, sensitive and specific detection of nucleic acids is continually emerging.
In this study, we presented the MCDA technology, as an isothermal amplification technique, which was carried out at a constant temperature (61-65 °C). Thus, MCDA did not require thermal denaturation of templates, and eliminated the use of the temperature-regulating equipment. Therefore, the MCDA can  be as a potential tool for developing hand-held diagnostic devices, which can be used to detect pathogens and diagnose clinical disease in research on environment hygiene, point-of-care diagnosis, 'on-site' testing and more.
Achieving efficient genetic analysis with the MCDA assay relies not only on the performance of nucleic acids amplification, but is also dependent on the technique selected for monitoring the MCDA reaction 7 . The FD colorimetric indicator, a simple visual detection method for the results of the MCDA  reaction, presents visual discrimination of the positive amplification within 40-min, alleviating the use of costly specialized equipment 9 . Moreover, the FD reagent allows for a single-step MCDA assay and the reaction tubes are not opened after the amplification, thus the risk of post-or cross-contamination is much lower 10 . Therefore, the MCDA technique combined with FD reagent should be useful in various fields, such as field diagnosis, point-of-care testing and more. Gel electrophoresis has been used as a mainstream method for detecting the amplicons of isothermal amplification approaches, even as a "gold standard" in many situations 11,12 . Hence, the technique was also employed to detect MCDA amplicons, and the positive reactions produced many bands of different sizes upon 2.5% agarose gel electrophoresis   because the MCDA products consist of various short segments of DNAs and stem-loop DNAs with different stem lengths (Fig. 3B). Moreover, the results of the MCDA amplification were monitored in real-time with optical instruments (turbidimeters), and the detection results were reported in a typical kinetics graph (Figs 5,7 and 11). The real time turbidity method did not require special indicators, probes or auxiliary regents except for the turbidimeter. Likewise, the risk of amplicon contamination was also obviated.
In the MCDA assay, a set of ten primers with ten binding sites, which hybridizes correctly to target sequences, provides a high degree of specificity, and the detection specificity was verified in our study. The primers in the MCDA assay only amplified the genomic DNA from L. monocytogenes strains and did not amplify the templates from the non-L. monocytogenens strain and negative control (Fig. 9). The results demonstrated that the novel MCDA technique offered high selectivity for analyzing the target sequences.
The newly established MCDA assay detected as little as 62.5 fg DNA per reaction and was at least 160-fold more sensitive than that of PCR, which has been report for L. monocytogenes detection [13][14][15] . Due to reduce the need for sophisticated equipment, have short turnaround times and low running costs, the MCDA assay is more suitable than PCR for simple, sensitive and rapid detection in various fields. Comparing with other isothermal amplification approaches (such as LAMP, CPA), MCDA was 16-and 32-fold more sensitive than the LAMP and CPA assays, respectively, and the positive reactions were obtained in as short as 15 minutes (Table 2, Fig. 7A). Thus, our assay has advantages over other isothermal techniques, namely high sensitivity and quick results, and is a good alternative to these methods for detection of trace amounts of target sequences in various samples.
In order to determine the practical application of MCDA detection to L. monocytogenes in food samples, 55 pork samples were analyzed by PCR, conventional culture-biotechnical detection and MCDA methods. Six pork samples were positive for L. monocytogenes using the MCDA and culture-biotechnical assays, but negative by PCR. Thus, the MCDA offered higher analysis sensitivity for the practical application compared to PCR. The poor sensitivity of PCR may be due to the reasons that the presence of non-target DNA in samples and some inhibitors (such as serum, blood and food ingredients) specific to the PCR affected the reaction sensitivity, or the copy numbers of the target templates were lower than the limit of detection [16][17][18] . Thereby, MCDA method is more tolerant to PCR inhibitors in samples and does not appear to be affected by presence of non-target sequences. Moreover, our study verified 100% agreement in L. monocytognenes-specific detection using MCDA and culture-biotechnical methods, while the MCDA assay was less expensive and timesaving.
The MCDA was formulated and developed as an isothermal amplification with the requirement for a set of 10 primers, which recognized 10 distinct regions of target sequences. In this manifestation, the MCDA technology has been verified as a rapid, highly sensitive and specific analysis tool for a target nucleic acid sequence. The primer design for MCDA technique requires the selection of 10 separate binding sites, and the CP1 and CP2 primers, containing at 5' end of the sequence complementary to C1 and C2, presented the restrictions on their positioning respective to each other. As a sequence, the primer design for MCDA assay is a disadvantage for its more widespread application, when targeting sequences containing complex secondary structure, shorter specific regions or highly polymorphic markers. For this case, S-MCDA technology may be another option.
As an alternative to MCDA assay, the S-MCDA also achieves highly analytical specificity and this is due to the fact that a set of 7 primers for S-MCDA spanning 8 distinct regions of the target sequence was designed. The analytical sensitivity of L. monocytogenes S-MCDA method was 125 fg genomic DNA per test and 80-fold more sensitive than PCR, and the specificity was 100% 13,15 . What's more, the S-MCDA technology provides a wider range of options for primer design since several of those primers can be positioned with fewer restrictions. For the S 1 -MCDA (S 2 -MCDA) assay, R1, CP2 and F2 (R2, CP1 and F1) primers can be designed with fewer restrictions, and the binding sites were not orientated in several particular regions.

Conclusions
A novel nucleic acid amplified detection method was established on the basis of isothermal strand-displacement polymerization reaction. The MCDA assay developed here was rapid, robust, specific and sensitive, which had the advantages over the conventional PCR technique, namely, modest equipment requirements, easy operation, quick results, cost and energy efficiency, and high sensitivity and specificity. Moreover, the novel technology eliminated the thermal cycle steps (an isothermal amplification at a single temperature), and incubating in a short time (40-min for MCDA, 50-min for S-MCDA) using a simple block heater or water bath was sufficient to replicate nucleic acids to detectable levels. Herein, these attractive traits will motivate the researchers to explore the application of the novel MCDA technique for nucleic acid analysis in various fields.

Bacterial strains.
A total of 90 bacterial strains, including 40 non-L. monocytogenes and 50 L. monocytogenes strains, were used for the exclusivity and inclusivity tests, respectively (Table 3). All Listeria and other non-Listeria strains were cultured on brain heart infusion plates overnight at 37 °C. Strain EGD-e was used as a positive control to test the appropriate conditions for MCDA and to establish baseline specificity and sensitivity.
Genomic DNA extraction. Bacterial  Three mainstream methods were used to monitor the MCDA amplification. The color change of positive MCDA reactions could be directly observed by FD reagent, and the amplified products were also detected by electrophoresis on 2.5% agarose gels with ethidium bromide staining. Furthermore, real-time measurement of MCDA reactions was performed by recording the optical density (OD) at 650 nm every 6 s using the Loopamp Real-time Turbidimeter LA-320C (Eiken Chemical Co., Ltd, Japan). A positive amplification was defined as a threshold value of > 0.1 and analysis of each sample (dilution) was tested at least two times.
In order to determine the optimal amplification temperature, the reaction mixtures of MCDA were incubated at a constant temperature ranging from 60 °C to 67 °C for 1 h and then heated at 95 °C for 5 min to stop the reaction. Mixture without DNA template was used as a negative control.

Validation by sequencing.
To further confirm the reliability and specificity of the MCDA assay, a MCDA-PCR-sequencing strategy was applied to analyze the MCDA products. The MCDA products corresponding to the level of 62.5 fg template DNA were verified by agarose gel electrophoresis, and the ladders between 100 bps and 1500 bps were extracted and purified by using QIAquick Gel Extraction Kit (QIAGEN, Hidlen, Germany) to obtain the template DNA for PCR. A set of two primers (P1 and P2) was used (Table 1)    template for sequencing. Sequencing was performed by Tsingke (Beijing, China) and the sequence data were compared with the target gene sequences in GenBank database.
Analytical sensitivity of the MCDA assays. To test the limit of detection (LoD) of MCDA and S-MCDA approaches, amplification reactions were performed using serial dilutions (2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 125 fg, 62.5 fg and 31.25 fg per microliter) of pure L. monocytogenes genomic DNA (strain EGD-e). 1 μ l DNA template was added into the reaction mixtures and three replicates of each dilution were examined to assess the sensitivity of MCDA and S-MCDA assays.
To compare the sensitivity of the MCDA, LAMP and CPA assays in pure culture, template DNA from strain EGD-e was serially diluted (2.5 ng, 250 pg, 25 pg, 2.5 pg, 2 pg, 1.5 pg, 1 pg, 500 fg 250 fg 125 fg per microliter), which were used for accurately determining the LoD of LAMP and CPA approaches. The LoD of LAMP and CPA technologies were ascertained by genomic DNA amount of the template. The hlyA-LAMP method has been developed by Wang L et al. and the L. monocytogenes-CPA assay has been also established by Wang Y et al., which were employed to confirm the LoD of LAMP and CPA methodologies 13,15 . Analytical specificity of the MCDA assays. To evaluate the specificity of the MCDA approaches, the MCDA reactions were carried out under the optimal conditions tested above with genomic DNA templates from the 40 non L. monocytogenes and 50 L. monocytogenes strains ( Table 3). Analysis of each sample was tested at least two times.