Detection of Salmonella enterica subsp. enterica via Quenching of Unincorporated Amplification Signal Reporters in Loop-Mediated Isothermal Amplification

Salmonella enterica is a major cause of diarrheal diseases in developing countries where timely surveillance and proper clinical management are inadequate. In this study, a rapid and cheap method of detecting S. enterica DNA was developed by employing the Quenching of Unincorporated Amplification Signal Reporters in Loop-Mediated Isothermal Amplification (QUASR LAMP) platform. QUASR LAMP provides a closed-tube, target-specific endpoint detection of pathogens, wherein results can be analyzed visually through an LED transilluminator and verified through agarose gel electrophoresis. Based on the chromosomal SopD gene, primers and probes were first designed, then screened. The assay was subsequently optimized so that the presence of S. enterica is determined by incubating the extracted DNA at 65°C for only 60 minutes. The assay was repeatable and can be performed by simply using a thermal cycler or even a dry bath incubator. S. enterica positives appear bright yellow green when viewed through a yellow filter excited with blue LED. The developed assay had an in silico and in vitro specificity towards Salmonella enterica subsp. enterica serovars with a limit of detection of 104 copies per microliter. The Salmonella QUASR LAMP assay has the potential for food and environmental applications. Chiefly, as an alternative to traditional microbiology and PCR, this QUASR LAMP assay can be used for point-of-care salmonellosis testing of clinical specimens in low-resource settings.


Introduction
Salmonella enterica is one of the most common causes of food-borne illnesses in developed countries and the major cause of diarrheal diseases in developing countries [1]. Aside from being the infectious agent of typhoid fever, S. enterica is also known to cause bacterial food poisoning. Nontyphoidal Salmonella (NTS) causes self-limiting diarrhea in an immunocompetent host. For an immunocompromised host, it can develop into a systemic disease. NTS infections are a major global threat, aficting an estimated ninety-three million people annually worldwide [2]. Salmonellosis in humans is generally contracted through the consumption of contaminated foods of animal origin (i.e., eggs, meat, poultry, and milk), although other foods, including green vegetables contaminated by manure, have been implicated in its transmission [3]. Terefore, surveillance systems on foodborne diseases such as salmonellosis are necessary to detect and respond to enteric infections in their early stages and thus prevent them from further spreading.
S. enterica possesses many virulence strategies employed to interact with the host defense mechanisms. Te SopD efector protein, encoded by the chromosomal SopD gene, presumably increases infammation and fuid secretion during gastroenteritis by directly promoting Salmonella invasion [1]. SopD also plays a role during desiccation survival which makes it survive several weeks in a dry environment and several months in water [4,5]. Unfortunately, current diagnostics for Salmonella infections, including invasive NTS, are inadequate to guide timely surveillance and proper clinical management [6,7].
Aside from the standard culture methods, nucleic acid amplifcation tests (NAATs) can be used to detect DNA in biological samples to detect contamination and diagnose pathogenic infection from S. enterica [8]. NAATs are used as a staple molecular diagnostic for infectious diseases [9][10][11]. Common NAATs include conventional polymerase chain reaction (PCR) and quantitative PCR (qPCR). qPCR provides great sensitivity and precision [12]. However, it requires a well-equipped laboratory and, thus, poses a major challenge for point-of-care applications. Moreover, qPCR requires highly purifed extracted nucleic acid, cold-chain reagents, and nonportable instrumentation that usually demands high electrical power [10].
In contrast, loop-mediated isothermal amplifcation (LAMP), an isothermal nucleic acid amplifcation technique, ofers a useful alternative to PCR for low-cost diagnostics for infectious diseases (Figure 1) [13]. Tis technique utilizes primer-based amplifcation of DNA and RNA targets (Figure 2(a)) [14]. Tis type of NAAT is robust and sensitive and can work with minimal or no sample treatment. However, the available detection techniques used in LAMP are not easily amenable to multiplexing to distinguish multiple targets in a single reaction [15]. LAMP results are typically analyzed by running the product on a gel [16] or by adding a dye post-reaction [17,18], which requires opening the tube after amplifcation and presents a risk for amplicon contamination ( Figure 1). Tus, most of LAMP's detection mechanisms are nonspecifc and prone to false positives.
A novel approach for the endpoint determination of LAMP is based upon the quenching of unincorporated amplifcation signal reporters (QUASR) [19]. In QUASR, one of the primers in the LAMP reaction is labeled with a dye (Figure 3). Compared to traditional LAMP, wherein detection is nonspecifc through the addition of a dye, QUASR LAMP ofers sequence-specifc detection because the fuorescent dye is already appended to the primer. Additionally, the reaction mixture contains a short probe, labeled with a dark quencher at the 3′ end, which is complementary to 7-13 bases at the 5′ end of the dye-labeled primer. Te quench probe is present in slight excess relative to the labeled primer and has Tm > 10°C below the temperature of the LAMP reaction so that it remains dissociated during the amplifcation. After incubation, the reaction is cooled to ambient temperature, resulting in dark quenching of fuorescent primers (i.e., negative reactions), or highly fuorescent amplicons (i.e., positive reactions) [19]. By combining multiple QUASR LAMP primer sets specifc for diferent targets, spectrally multiplexed detection can be achieved [19]. QUASR is reported to provide a closed-tube, target-specifc endpoint detection ( Figure 4). It has large discrimination between positive and negative samples and requires minimal instrumentation [19][20][21]. Terefore, QUASR can be suitable for deployable LAMP detection of Salmonella enterica. Tis study, therefore, aimed to demonstrate the feasibility of the QUASR LAMP assay in detecting Salmonella enterica in bacterial samples.

LAMP Primer Design.
Te SopD gene was targeted for detecting general Salmonella enterica subsp. enterica. Available Salmonella enterica subsp. enterica SopD gene sequences were obtained from the GenBank database. Primer Explorer v.5 software (Fujitsu Ltd. 2015) was used with default parameters to scan for suitable LAMP primer sets for the SopD gene (GenBank NZ_CP009102.1). Four types of LAMP primers were designed using Primer Explorer v.5 (Fujitsu Ltd, 2015) based on the following six distinct regions of the target gene, designated in Figure 2(b) on the right from the 5′-end as F3, F2, F1, B1, B2, and B3 (Table 1). Te forward inner primer (FIP) consists of the F2 sequence (at its 3′ end) that is complementary to the F2c region and the same sequence as the F1c region at its 5′ end [16].

LAMP Primer Screening and Preliminary Assay.
Before proceeding to QUASR LAMP optimization, the SopD primers were initially screened by LAMP. Te assays were (1) A clinical sample (e.g., blood, serum, stool, nasal swab) from the patient is frst collected and then subjected to (2) nucleic acid extraction, where the DNA or RNA from the sample is isolated, purifed, and concentrated. (3) Te extracted nucleic acid is then added to the LAMP reaction mixture and incubated isothermally for 60 minutes. (4) After amplifcation, the reaction tube is opened, and an intercalating dye (SYBR green dye) is put into the mixture. (5) Analysis can be done using the naked eye or by viewing through a blue LED light. Using the naked eye, positives appear yellowgreen, and negatives appear orange. Trough a blue LED light, positives appear as bright fuorescent yellow green, and negatives appear as dull and colorless. Agarose gel electrophoresis can also be performed to confrm the amplifcation of target products. Ladder-like bands are characteristic of LAMP products.   One of the loop primers (LF or LB) or inner primers (FIP or BIP) is labeled with a dye. Te reaction mixture also contains a short probe, labeled with a dark quencher at the 3′ end and complementary to 7-13 bases at the 5′ end of the dye-labeled primer. Te quench probe is present in slight excess relative to the labeled primer and has Tm > 10°C below the temperature of the LAMP reaction, such that it remains dissociated during the amplifcation. After incubation, the reaction is cooled to ambient temperature, resulting in the dark quenching of fuorescent primers (negative reactions) or highly fuorescent amplicons (positive reactions) [16].  Reaction tubes were incubated at 60°C for 50 minutes, then stopped at 80°C for 2 minutes. LAMP results were analyzed visually using the naked eye and a blue-light LED illuminator. Results were also verifed with agarose gel electrophoresis data showing the characteristic ladder-like patterns.

Fluorophore-Labeled Primer and Quencher Probe Design.
To design the fuorophore-labeled primers for the QUASR LAMP reaction, the 5′ end of the FIP primers was appended with a fuorophore ( Table 2). Te FAM reporter dye (green fuorophore) was used for the SopD FIP. A short quencher probe complementary to the 5′ end of the labeled primer (FIPc) was manually designed. Te quencher probes were modifed at the 3′ ends with a dark quencher (e.g., Black Hole quencher [BHQ]). Te quencher used for SopD FIP is BHQ-1. Te designed quencher probes were analyzed such that they were not likely to form stable hairpins. Dye-labeled primers and quenching probes were ordered from Macrogen (Seoul, Korea).  (Table 3).

DNA Isolation.
Genomic DNA was extracted from bacterial cultures that were grown in Tryptic Soy Broth at 37°C for 24 h using the GenAmplifyTM DNA Purifcation Kit (Manila HealthTek, Inc., Marikina, Philippines) according to the manufacturer's instructions.

QUASR-LAMP Assay Optimization.
After confrming that the primers designed were useable and specifc to the target organism, the QUASR LAMP assay was optimized. Te designed fuorophore-labeled primers and quencher probes were incorporated into the reaction mixture. Optimization was done to obtain the result with the brightest  Te Scientifc World Journal 5 fuorescence in the positive control (PC) and the least background fuorescence in the no template control (NTC). All QUASR LAMP assays were done with 12.5 μL reaction volumes in clear 0.2 mL PCR tubes with fat caps. Figure 4(b) summarizes the QUASR LAMP assay interpretation for the target. S. enterica positives appear bright yellow green. All results were verifed with gel electrophoresis data wherein positives show the characteristic ladder-like patterns. Te initial QUASR-LAMP formula used was based on a previously established functional formula for detecting other organisms. Te assays were performed with 2.5 μL sample DNA as a template in a total volume of 12. From this formula, the optimal incubation temperature was determined by performing gradient QUASR LAMP in a T100 Termal Cycler (Bio-Rad Laboratories, Inc., CA, USA). Te following protocol was used for gradient QUASR LAMP assays: amplifcation at 60-65°C for 50 min, stop reaction and Bst polymerase inactivation at 80°C for 2 min, then cool down at 4°C for a minimum of 5 min.
To determine the optimal amplifcation time for the reaction, diferent incubation periods (30, 40, 50, and 60 min) were evaluated. For the detection of S. enterica using the SopD primer set, the following was the optimized protocol which was used for subsequent tests: amplifcation at 65°C for 60 min, stop reaction and Bst polymerase inactivation at 80°C for 2 min, then cool down at 4°C for a minimum of 5 min. Reactions were incubated using a T100 Termal Cycler (Bio-Rad Laboratories, Inc., CA, USA).

Repeatability.
Repeatability expresses the precision under the same operating conditions over a short interval of time [23]. Tree diferent assays were done by one operator over three days using three replicates of the no template control (NTC), the 10 6 cp/μL positive control (PC), and the S. enterica sv. Typhi DNA sample. Repeatability assays were performed using two setups, one using a T100 Termal Cycler (Bio-Rad Laboratories, Inc., CA, USA) and another using a lab-in-a-mug dry bath incubator (Manila HealthTek, Inc., Philippines).

Analytical Characteristics.
To confrm that the acquired SopD primer set would indeed amplify the target organism of interest, the outermost primers (F3 and B3) were subjected to in silico amplifcation. In silico PCR was performed using a freely available molecular biology resource for experiments against prokaryotic genomes (http://insilico.ehu.es/). Te F3 and B3 primer sequences were input, and in silico PCR amplifcation was done against diferent bacterial genomes. Te optimized QUASR LAMP assay was also tested in vitro against the positive and negative control panels listed in Table 3. Furthermore, the analytical sensitivity was determined based on three replicates of ten sample concentrations (10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , and 10 0 copies of DNA per microliter (cp/μL)) of an S. enterica sv. Typhi DNA sample.

QUASR-LAMP Assay Optimization.
Te key factors for LAMP primer design are melting temperature (Tm), stability at the 3′ and 5′ ends of each primer (ΔG), % guanine and cytosine (GC) content (optimal GC content range between 40% and 60%), and secondary structures. Moreover, since multiple primers anneal in diferent target regions at the same time, it is essential to consider the distance between primers and primer secondary structures such as self-dimers and cross dimers [22].
In designing the LAMP primers for this study, the Tm for each region was intended to be about 65°C (64-66°C) for F1c and B1c, and about 60°C (59-61°C) for F2, B2, F3, and B3. Te 3′ ends of F2/B2 and F3/B3, and the 5′ end of F1c/B1c were designed so that the free energy (ΔG) is -4 kcal/mol or less. Primers were also designed so that their GC content is between 40% and 65%. To prevent the formation of primer dimers, it was ensured that the 3′ ends were not complementary. Te distance from the end of F2 to the end of B2 (the region amplifed by the LAMP method) is between 120 and 160 bases; the distance from the 5′ end of F2 to the 5′ end of F1 (the portion that forms the loop) is between 40 and 60 bases; and the distance between F2 and F3 is between 0 and 60 bases (A Guide to LAMP primer designing (PrimerExplorer V4). Eiken Chemical Co., Ltd.). Considering these key factors, the SopD primer set (Table 1) passed the initial LAMP screening and was used for further QUASR LAMP optimization.
LAMP is based on auto-cyclingstrand-displacement DNA synthesis carried out by Bst DNA polymerase large fragment under isothermal conditions [24]. Additionally, Bst DNA polymerase, an enzyme derived from Geobacillus Table 2: SopD QUASR LAMP fuorophore-labeled primer and quencher probe set. Te 5′ end of the FIP primer was appended with the FAM reporter dye (a green fuorophore). A short quencher probe complementary to the 5′ end of the labeled primer (FIPc) was manually designed. Te quencher probe was modifed at the 3′ end with a dark quencher (e.g., Black Hole quencher (BHQ)).

Label
Tm (  stearothermophilus, has an optimal temperature ranging from 60°C to 65°C for general strand-displacement reactions and a deactivation temperature of 80°C [25]. As in PCR, factors such as incubation temperature and time are crucial for the in vitro amplifcation of nucleic acid targets in QUASR LAMP. Te optimal incubation temperature for the assay was determined by performing gradient QUASR LAMP in a T100 Termal Cycler using six incubation temperatures: 60°C, 61°C, 62°C, 63°C, 64°C, and 65°C. Tough the desired results were seen in the 60°C, 62°C, and 63°C setups ( Figure 5), wherein negative reaction tubes (NTCs) appear dull and nonfuorescent while positive reaction tubes (PCs) appear bright fuorescent green, agarose gel verifcation data implies otherwise. In these temperatures, some of the NTC tubes showed ladder-like bands in 1.5% agarose gel which is characteristic of positive reactions. Moreover, at the 61°C and 64°C temperature points, one out of three NTC tubes showed a positive reaction in both the LED transilluminator and 1.5% AGE data. Terefore, the optimal temperature for the assay incubation is 65°C because NTC and PC reaction tubes yielded the expected results as seen in both the LED transilluminator and 1.5% AGE data ( Figure 5).
Te optimal duration of the assay incubation was also determined by running the assay for 30, 40, 50, and 60 minutes ( Figure 6). Te 30-minute incubation time did not produce good contrast between NTC and PC reaction tubes. One NTC tube from the 40-min incubation setup showed a false-positive reaction. At the 50-and 60-min incubation points, the NTC and PC reaction tubes yielded -/+ -/+ -+ + + + + + -/+ -/+ -+ + + + + -/+ + + + + -/+ --+ -+ + + + + + --- Figure 5: Optimization of assay incubation temperature. Sample (2.5 μL) was added to 10 μL reagent mix then incubated at 60-65°C for 60 minutes, 80°C for 2 minutes, and cooled to 4°C for 10 minutes using a T100 Termal Cycler (S/N 621BR14432). Samples used were the No Template Control (NTC) and the Positive Control (PC; 10 6 copies/μL). Results were viewed using the EasyView ™ LED Transilluminator (Manila HealthTek, Inc., Marikina, Philippines) which has a blue LED light and a yellow plastic gel flter, and through agarose gel electrophoresis (1.5% agarose, 135 V for 25 minutes). Salmonella enterica subsp. enterica positives appear bright yellow green. All images were taken using a Canon EOS 750D camera. Interpretation of results are as follows: (+)-Positive using the LED transilluminator and positive in 1.5% AGE. (−)-Negative using the LED transilluminator and negative in 1.5% AGE. (−/+)-Negative using the LED transilluminator but positive in 1.5% AGE. 8 Te Scientifc World Journal    the expected results, as seen in both the LED transilluminator and 1.5% AGE data. Tus, the optimal incubation time ranges from 50 to 60 minutes. Te 60-min incubation period was selected for subsequent experiments since low input or inhibitor-containing samples may require extended incubation times to visualize detection (New England Biolabs, Inc., https://www.neb.sg). From this point, all Salmonella QUASR LAMP assays were performed by incubating the reaction tubes at 65°C for 60 minutes, followed by 80°C for 2 minutes, then 4°C for 10 minutes. Te Bst polymerase is generally deactivated at 80°C. Furthermore, cooling down at 4°C provides a chance for unused fuorescent primers to be quenched and prevents false positive detection.

Repeatability.
Repeatability expresses the precision under the same operating conditions over a short interval of time [23]. To confrm if the assay can be performed using a simple dry bath incubator, repeatability assays were performed using two setups, one using a conventional thermal cycler and the other using a dry bath incubator.
As seen in Figure 7, the Salmonella QUASR LAMP assay was repeatable for both setups.
Consistently for all replicates over three runs, the NTC reaction tubes did not show amplifcation and the PC reaction tubes showed positive amplifcation as seen in both the LED transilluminator and 1.5% AGE data for the two setups. Tus, there was an agreement between replicates within and between assay runs by the same operator over  Figure 9: Assay sensitivity. Sample (2.5 μL) was added to 10 µL reagent mix then incubated at 65°C for 60 minutes, 80°C for 2 minutes, and cooled to 4°C for 10 minutes using T100 Termal Cycler (S/N 621BR14432; Bio-Rad Laboratories, Inc., CA, USA). Samples used were the No Template Control (NTC), Salmonella enterica subsp. enterica sv. Typhi DNA (STY), and Positive Control (PC). Te following concentrations of PC were used in triplicate: 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , and 10 0 copies per μL. Results were viewed using the EasyView ™ LED Transilluminator (Manila HealthTek, Inc., Marikina, Philippines) which has a blue LED light and a yellow plastic gel flter, and through agarose gel electrophoresis (1.5% agarose, 135 V for 25 minutes). Salmonella enterica subsp. enterica positives appear bright yellow-green. All images were taken using a Canon EOS 750D camera. Interpretation of results are as follows: (+)-Positive using the LED transilluminator and positive in 1.5% AGE; (−)-Negative using the LED transilluminator and negative in 1.5% AGE. a short period of time [23]. Tis also underscores the simplicity of QUASR LAMP machinery, wherein it can be performed by laboratories with either a T100 Termal Cycler or a dry bath incubator. As compared to the machine and power requirements of a real-time PCR, the testing laboratory in this scenario can thus cut down on expenses, especially in locations where stable electricity and budgetary allocations are limited.

Analytical Characteristics.
To test the ability of the assay to exclusively identify the intended target, the analytical specifcity was tested in silico and in vitro. Te results of in silico PCR amplifcation using the online tool (http://insilico. ehu.es/) are shown in Table 4. Using the SopD outer primers (F3 and B3), positive amplifcation (indicated by the plus sign) was observed for all Salmonella enterica subsp. enterica serovars ( Table 4). As expected, the designed primers showed no cross-reactivity with other bacterial genomes (indicated by the minus sign, Table 4).
Te results of in vitro specifcity testing showed that the assay could specifcally amplify Salmonella enterica subsp. enterica serovars Typhi, Paratyphi A, Paratyphi B, and Typhimurium ( Figure 8). No amplifcation was observed in the negative control panel samples. Hence, there was an agreement between the in silico and in vitro tests using the available control panels.
In this study, the analytical sensitivity was determined based on three replicates of nine sample concentrations (copies per μL, cp/μL) of the positive control. Te Salmonella QUASR LAMP assay was able to detect down to 10 4 cp/μL of DNA target, in three out of three replicates (Figure 9).
Salmonella spp. can be detected in humans by testing clinical specimens such as blood and stool. Since PCR and QUASR LAMP are both molecular assays, the required volume for testing is minimal (200-500 μL for blood and >1 g for stool). A study using real-time PCR amplifcation for the detection of invasive Salmonella serovars showed better limit of detection (LOD) values [26]. Te number of copies of target DNA detected for S. Paratyphi A was about thirty-nine copies per mL of blood. Te S. Typhi positive amplifcations showed a LOD of about sixty copies per mL of whole blood. Te analytical sensitivity of the developed Salmonella QUASR LAMP assay can be further improved to be up to par with rt-PCR values. Trough the inclusion of detergent additives such as Triton X-100 and dimethyl sulfoxide (DMSO) in the reagent mixture, the analytical sensitivity of the assay can be enhanced [27].

Conclusions and Recommendations
Quenching of unincorporated amplifcation signal reporters in loop-mediated isothermal amplifcation (QUASR LAMP) has been demonstrated to be useful in detecting Salmonella enterica subsp. enterica DNA. Tis study verifed that QUASR LAMP provides a closed-tube, target-specifc endpoint for visual detection. It has discrimination between positive and negative samples and requires minimal instrumentation.
Te developing Salmonella QUASR LAMP assay has applications in testing food, environmental, and clinical samples for the presence of Salmonella enterica subsp. enterica. Rapid, sensitive, and specifc detection of Salmonella enterica infections may lead to proper clinical management and better outcomes. Furthermore, the QUASR LAMP assay takes only about 60 minutes for a test, while PCR assays usually take 80 to 90 minutes, and culture techniques can take days before getting conclusive results. Tus, the developed assay can be used in low-resource settings as an alternative to PCR and traditional microbiological techniques.
Additional studies are being done to improve the current limit of detection of the assay (10 4 cp/μL) through the addition of detergents like DMSO and Triton X-100. QUASR-LAMP detection can also be improved by using an algorithm that utilizes chromaticity to analyze the fuorescence signal (e.g., phone or web applications). Analytical specifcity data can also be strengthened by testing a wider panel of positive controls (i.e., other Salmonella enterica subsp. enterica serovars) and negative controls (i.e., other closely related organisms). Te assay can also be further developed and optimized to perform multiplex detection of diferent Salmonella enterica subsp. enterica serovars or other closely related enteric bacteria and viruses. QUASR LAMP, coupled with a direct rapid method for nucleic acid isolation, can further improve the assay setup by minimizing the complexity of sample preparation and reducing the time of analysis. Te applicability of the developed assay can be determined by testing clinical specimens such as blood and stool, as well as by testing food and environmental samples.

Data Availability
Te data used to support the fndings of this study are included within the article.

Disclosure
Tis manuscript, its part or in full, has not been previously published and is not under copyright at another publication (print, online, or in a non-English journal).