Bioprocessing and integration of a high flux screening systematic platform based on isothermal amplification for the detection on 8 common pathogens

During a large variety of common pathogens, E. coli, P. aeruginosa, MRSA, MRCNS, V. parahaemolyticus, L. monocytogenes and Salmonella are the leading pathogens responsible for large number of human infections and diseases. In this study, a high flux screening based on nucleic acid isothermal amplification technique has been developed. For the 8 common pathogens, species-specific targets had been selected and analyzed for their unique specificity. After optimization, separate LAMP reaction assays had been bioprocessed and integrated into one systematic detection platform, including 8 strips (PCR tubes) and 96-well plates. Eight standard strains verified for the accuracy. Application of the established high flux screening platform was used for detection for 48 samples in 4 different 96-well plates, with 2 groups of 2 operators using double-blind procedure. The accuracy of 100% was obtained, with the total time consumption as 66–75 min (for 12 samples detection on 8 different pathogens). As concluded, through the bioprocess of the systematic platform based on LAMP technique, it’s been demonstrated to be capable of simultaneous detection of 8 pathogens, with high sensitivity, specificity, rapidity and convenience.

In recent years, PCR and Q-PCR have been well studied and documented to be a promising technique for rapid detection and bacterial identification [21][22][23][24][25]. However, regular PCR requires laborious results determination process, such as electrophoresis or hybridization. (The former takes several hours and the latter takes up to 36 h.) For Q-PCR, expensive equipment and reagents are required, which poses an obstacle during its broad application [26,27]. Since 2000, a novel nucleic acid amplification method has been developed and established, named loop-mediated isothermal amplification (LAMP) [28,29]. For the past 2 decades, LAMP has been developed for the detection of various microorganisms and other genes [30,31]. However, the clinical application of LAMP assays, especially 1 3 platform for high flux screening based on LAMP, has been rarely reported [32][33][34]. Advantages of LAMP include high sensitivity, specificity, rapidity, simpliness in operation and labor, convenience and expense [35]. However, lack of the bioprocessing and systematic integration of this technique into applicable platforms has been the biggest obstacle during the application of this technique [36-38]. As a consequence, development and application of rapid detection on such pathogens as well as their virulent factors for high flux screening application are of utmost importance and necessity [39][40][41].
In this study, a high flux screening of pathogens, including E. coli, P. aeruginosa, MRSA, MRCNS, V. parahaemolyticus, L. monocytogenes and Salmonella, has been developed based on the loop-mediated isothermal amplification methodology.

Design on the systematic integration of detection
Specific targets were selected for different pathogens, with the detailed information as follows. For E. coli, rfbE (the specific O-antigen) was selected. For P. aeruginosa, oprI was selected. For MRSA, MSSA, MRCNS, MSCNS, mecA, femA and 16SrRNA (specific for the genus of Staphylococci) were selected. For Salmonella, invA was selected. For V. parahaemolyticus, tlh (thermolable haemolysin, considered to be a species-specific marker for V. parahaemolyticus) gene was selected. For L. monocytogenes, hlyA was selected. In our previous studies, the primer sets for each of the targets had been separately designed, and optimized for LAMP reaction. Such primer sets were also selected in this study, and additionally, new primer sets for each targets had been designed using Primer Explorer V4 [42][43][44]. Optimal parameters were selected by each set of primers. Principles for the systematic integration of the high flux screening platform included: firstly, unique temperature is required for the reaction occurred for each primers set. Secondly, unique reaction time is also required for each target. The DNA samples of E. coli, Pseudomonas aeruginosa, MRSA, MSSA, MRCNS, MSCNS, L. monocytogenes, Salmonella, V. parahaemolyticus were isolated using the DNA extraction kit. DNA quality and concentration had been confirmed with NanoDrop before further detection. LAMP reaction was performed under different temperatures (59 °C [45]. The results determination was performed by observation by naked eye and SYBR Green I, electrophoresis [46,47]. At last, 8 primer sets were selected for the high flux screening platform (Table 1).

Bioprocessing the separate LAMP assays into high flux screening platform
As mentioned above, 8 primers sets specifically targeted for rfbE, oprI, mecA, femA, 16S rRNA for staphylococci, invA, tlh and hlyA have been included [48]. As LAMP reactions were concerned, 65 °C was selected due to the efficiency, stability and reproducibility of this methodology. Also, reaction time as 45 min was selected as at this time point sufficient reaction products for results determination as well as minimal amounts of amplicons were both achieved [49][50][51]. For the concentration of betaine, insignificant difference was found, and thus 0.3 M was used for the consideration of minimal expense. For the ratio between calcein and Mn 2+ , 1:4 was found to be optimal and thus selected. In addition, calcein was selected to replace SYBR Green I for the color change for results determination as calcein is capable of preload in the reaction volume but SYBR Green I [52]. For application, 8 strips (PCR tubes) were employed for the detection of standard strains, and 96-well plates were employed for the detection of different samples. Different sets of primers as well as reaction volume (25 micro liters were used, with 0.3 M betaine and 1:4 of calcein and Mn 2+ ) were prepared at each tube of 8 strips or 96-well plates (Fig. 1

Application of the high flux screening platform
In this study, a common type of food sample, cake was employed to verify the applicability of this high flux screening platform. Sample artificially contamination was

Development of separate LAMP platform and their integration
According to the development of LAMP assays, positive results had been obtained from DNA amplification of standard strains, with color change from orange to green by either SYBR Green I or calcein, as well as typical ladder bands pattern from electrophoresis [61][62][63]. Optimization of LAMP reaction was also performed, in details as follows. For reaction processed under different temperatures ranging  TGA CAA TCG CTT CTC AT  20  B3  GTT CTT CGC TTT GGC AAT GT  20  FIP  CTG TCA CCG AGT GCA ACC ACT TAA CCA CAC GAT CTG GAGCA  41  BIP  GCA TCA CAA TGG CGC TTC CCA CCG TTG GAG AAG TGA CCTA  40  LF  GTT GAT TTG ATC TGG CTG CATTG  23  LB AAC CCG AAC AGC TGG TTC T 19    (Fig. 2). In this study, calcein was selected to replace SYBR Green I for the color change for results determination as calcein is capable of preload in the reaction volume but SYBR Green I [64,65]. For different ratios of calcein and Mn2 + (1:20, 1:16, 1:12, 1:8, 1:4, 1:2), 1:4 was found to be optimal and thus selected (Fig. 3).  [66]. As shown, color change from orange to green was observed for positive results, and 100% of specificity was obtained in this study (Fig. 4a). From template DNA loading to results determination, 50 min was required (Fig. 4b).

Application of the high flux screening platform
According to the results, for the first round as Group A for strains selection and Group B for detection, Operator 1 had selected 1 E. coli, 1 P. aeruginosa, 2 MRSA,  [70]. In summary, 100% of accuracy was obtained by both groups for 2 rounds of experiments (Fig. 5). As rapidity was concerned, 10-15, 8-10, 45 and 3-5 min are required for DNA extraction, template DNA loading, LAMP reaction and results determination, respectively [71,72]. In summary, the total time consumption is 66-75 min, for simultaneous detection of 12 samples for 8 different pathogens.

Discussion
In this study, a high flux screening based on nucleic acid isothermal amplification technique has been developed. Firstly, 8 common pathogens were selected and 8 speciesspecific targets had been selected and analyzed for their unique specificity. Then, 8 different sets of primers for LAMP reaction had been further designed and optimized to obtain unique reaction temperature and time. Furtherly, the 8 detection assays had been integrated into a biosystem panel for isothermal detection, including 8 strips (PCR tubes) and 96-well plates, with 8 standard strains verified for the accuracy [73][74][75][76]. At last, application of the established high flux screening platform was used for detection for 48 samples in 4 different 96-well plates, with 2 groups of 2 operators using double-blind procedure. The accuracy of 100% was obtained, with the total time consumption as 66-75 min (for 12 samples detection on 8 different pathogens) [77][78][79]. As concluded, through the bioprocess of the systematic platform based on LAMP technique, it's been demonstrated to be capable of simultaneous detection of 8 pathogens, with high sensitivity, specificity, rapidity and convenience.
Acknowledgements This study was supported by Grants from the Science, Technology and Innovation Commission of Shenzhen municipality (JCYJ20170306113900538; JCYJ20170306140020487), and the Sanming Project of Medicine in Shenzhen (SZSM201812090).