1. Introduction
Early mortality syndrome (EMS), also named acute hepatopancreatic necrosis disease (AHPND), is an acute severe liver disease in Pacific white shrimps,
Litopenaeus vannamei. The mortality of the disease can be as high as 90% within 35 days in severely infected ponds [
1].
Vibrio parahaemolyticus (VP) bacteria, spreading from the gastrointestinal tract to hepatopancreas tissues, was identified as the cause of the disease. EMS was first discovered in 2010, spreading across shrimp ponds in Southern China, and later affecting the shrimp business throughout the Southeast Asia region, causing an estimated loss of more than one billion USD yearly since 2012 [
2].
The polymerase chain reaction (PCR) technique has been widely adopted for the early detection of pathogens in shrimp ponds [
3]. The PCR technique is well-known for its capacity for exponential DNA amplification by thermal cycling of DNA samples using two main reagents, primers and DNA polymerase. Coupled with gel electrophoresis techniques, either by agarose gel electrophoresis or polyacrylamide gel electrophoresis, the so-called “PCR method” requires about 30 to 40 rounds of thermal cycling, in which all processes must be carried out in a standard laboratory, and around one day to deliver the diagnosis results to the shrimp farms. Meanwhile, quantitative polymerase chain reaction (qPCR) is used for the quantitative measurement of a normalized reporter value (Rn value) to identify the specific signal generated from a given set of PCR conditions under close monitoring. The qPCR TaqMan probe was adopted for shrimp pond pathogen detection in laboratory bioassays, with a reported detection sensitivity of around 10
2 copies [
4].
The loop-mediated isothermal amplification method (LAMP) has been adopted as an alternative method for DNA amplification and detection, with less processing time and simpler procedures than the conventional PCR and qPCR methods. The LAMP method relies on Bst DNA/RNA polymerase enzymes, which react at isothermal temperatures [
5], thus eliminating the thermal cycling process required for typical PCR and qPCR methods. The LAMP method also allows visible detection, which is convenient for field test applications. For example, magnesium pyrophosphate can be used to induce the sedimentation of reaction products [
6], fluorescent substances can be introduced for ultraviolet (UV) light detection [
7], and simple methods such as the introduction of pH-sensitive dye can be applied to change the color of products for visible detection [
8].
Table 1 shows various examples of LAMP applications and the required quantities of reactants.
To encourage more frequent use of the LAMP method and to ensure that reliable detection results and insignificant contamination are achieved in field test applications, lab-on-a-chip platforms (LOC), relying on various microfluidic techniques, have been applied and can be found in a variety of studies [
14,
15,
16,
17,
18,
19,
20,
21,
22,
23].
Table 2 shows examples of successful applications of LOC platforms in chemical and biochemical detection. In this work, the so-called microfluid technique electrowetting-on-dielectric (EWOD)—sometimes known by its more common name, “digital microfluidics”—was selected for creating LAMP–LOC platforms for the field detection of EMS in shrimp ponds. Today, EWOD devices are commonly used for microdroplet manipulation, i.e., transporting, splitting, mixing, and dispensing [
24,
25,
26], and are quite suitable for manipulating the chemical reactants involved in LAMP detection, as mentioned in
Table 1 and
Table 2.
The required quantities of chemical reactants for LAMP detection are normally between 1 and 25 μL. To prevent contamination from the surroundings, the closed-typed EWOD platform is composed of eight layers, i.e., substrate, bottom electrodes, bottom dielectric, bottom hydrophobic layer, medium substance, top hydrophobic layer, top electrodes, and a transparent lid, as shown in
Figure 1, which shows the configuration of EWOD device selected for creating LAMP–LOC platforms. The working principle of EWOD devices is that, by applying an induced electrical field between two-adjacent electrodes on the bottom substrate to disturb the equilibrium of droplet surface tension, the electromotive force then causes the droplets to change their shape and move accordingly [
27], as shown in
Figure 2. In EWOD devices, droplet manipulation is performed on a flat plate and does not require a moving part, such as a micro-pump or any other complex mechanical configuration involving micro valves or micro piping networks; therefore, contamination resulting from assembly and operation can be avoided. Due to their ability to precisely manipulate liquid droplets combined with their compact configuration, EWOD devices are commonly used for proportioning chemical compounds in various reaction processes [
28].
In this work, a LAMP–LOC platform was created and proposed for utilization in the field detection of EMS in Pacific white shrimp ponds. The configuration of the LOC is clearly presented. The colorimetric LAMP assay with pH-sensitive xylenol orange (LAMP–XO), which allows visible observation detection throughout the process, was applied to create the proposed LAMP–LOC platform. The sensitivity of detection is validated by observing the limit of detection (LOD) of the LAMP–XO diagnosis technique performed on the proposed LAMP–LOC platform in comparison with the LOD of the standard LAMP laboratory tests.
2. Sample Preparation
To validate the detection sensitivity and accuracy of the proposed LAMP–LOC platform in comparison with the common LAMP laboratory tests for EMS detection, the chemical compounds must be prepared beforehand in a contamination-free laboratory, as shown in
Table 3. The purified EMS DNA samples were prepared following the recommendations made by Arunrut et al. [
29]. Four reference EMS DNA plasmids with four different concentrations at 10
2 copies, 10
3 copies, 10
4 copies and 10
8 copies were prepared by diluting the purified EMS DNA samples in sterile distilled water (SDW). LAMP reaction mixtures consisting of each reference EMS DNA plasmid, 0.2 µM each of forward outer primer (F3) and backward outer primer (B3), 2 µM each of forward loop primer (LF) and backward loop primer (LB), 2 µM each of forward inner primer (FIP) and backward inner primer (BIP), 1× buffer for LAMP dye (pH 8.5), 1.2 mM dNTPs (Thermo Fisher Scientific, Waltham, MA, USA), 6 mM MgSO
4 (New England Biolabs, Ipswich, England), 0.4 M betaine (Sigma Aldrich, St. Louis, MI, USA), 8 U Bst 2.0 WarmStart DNA Polymerase and 0.12 mM of pH-sensitive dye (xylenol orange; XO) (Sigma Aldrich, St. Louis, MI, USA) were prepared carefully. The volume of EMS DNA plasmids for standard laboratory tests was set at 2 μL, but due to the limit of the droplet size that could be handled by the LAMP–LOC platform with the proposed dimensions outlined in
Section 3.1, the droplet sizes of EMS DNA plasmids were limited to 3 μL for LAMP–LOC platform testing. All primers were bought from Bio Basic Inc., Canada, and the details of primer sequences are reported in
Table 4.
The LAMP reaction must be performed at the standard isothermal testing temperature of 64 °C for 75 min. Reaction mixtures without any DNA and with an EMS DNA plasmid concentration of 108 copies must be prepared prior to testing to serve as the negative control and the positive control for this validation test.
4. Qualitative Sensitivity Validation of LAMP–XO EWOD Platforms
Typically, the LAMP method was performed in a thermal cycler (Applied Biosystems™: SimpliAmp™ thermal cycler), in which thermal cycling for DNA/RNA amplification could be adjusted to provide isothermal DNA/RNA amplification according to the specified testing procedure. For EMS detection, 110-μL LAMP–XO premix and 5-μL Bst polymerase must be homogenously mixed in a vortex mixer and a centrifuge for 10 s. The mixed compound was then placed in 5 microtubes, each containing 23 μL of the substance. During the sensitivity validation test, carried out in order to observe the limit of detection (LOD), the 2-μL EMS DNA plasmid samples with concentrations of 0 copies, 10
2 copies, 10
3 copies, 10
4 copies and 10
8 copies were added to each microtube. The 0-copy EMS DNA plasmid sample was used as the negative reference and could be prepared using SDW. The microtubes were then placed into a vortex mixer and a centrifuge to guarantee homogenous conditions prior to being placed in a thermal cycler. In the thermal cycler, DNA/RNA amplification was maintained at 64 °C for 75 min. After the isothermal amplification process, the color of the positive samples supposedly turned from violet to yellow, as shown in
Figure 15. The sensitivity of EMS detection by the standard LAMP tests was limited to 10
2 copies, in agreement with the work carried out in [
29].
An experimental test for EMS detection on the LAMP–LOC platform was performed to observe the sensitivity and repeatability of the proposed LAMP–LOC platform. To prevent contamination on the LAMP–LOC parts that come into direct contact with the samples, a new and sterilized polytetrafluoroethylene (PTFE) film was applied to the bottom plate and the FTO top layer/lid was washed with AM9890 DNA Zap™ PCR DNA degradation solution. During the test, a homogenous mixture of 88-μL LAMP–XO premix and 4-μL Bst polymerase was equally divided and placed using a micropipette through the drilled holes at each mixing station, while 72 μL of DNA plasmid was placed at the inner dispensing station. The four different concentrations of EMS DNA plasmid samples selected, with concentrations of 0 copies, 10
2 copies, 10
3 copies and 10
8 copies, were used to validate the sensitivity of the LAMP–LOC platform. For each plasmid concentration, 3 μL plasmid samples were automatically and simultaneously dispensed and delivered to the four mixing stations, as shown on the top of
Figure 16, where the red lines envelop the boundary of the EMS DNA plasmid droplets dispensed and delivered to the mixing stations. After homogenous mixing conditions were achieved at each mixing station, the droplets at each mixing station underwent an isothermal heating process at 64 ± 1 °C for 75 min to provide sufficient DNA/RNA amplification for EMS detection. Xylenol orange (XO) causes the color of samples to change from violet to yellow in the event of
Vibrio parahaemolyticus (VP) infection. The experimental results show that the proposed LAMP–LOC platform can provide positive detection results at all four stations for plasmid concentrations of 10
2 copies, 10
3 copies and 10
8 copies, as shown on the bottom of
Figure 16, where the color change happens as early as 60 min for the high plasmid concentration of 10
8 copies, and the color change happens clearly after 75 min for the lower plasmid concentration of 10
2 copies and 10
3 copies under the isothermal amplification process. In
Table 6, the test results obtained using the standard LAMP–XO technique performed on a thermal cycler, and the LAMP–XO technique on the LAMP–LOC platform, were compared with the test results based on the standard LAMP procedure with gold nanoparticles as probes (LAMP–AuNP) and the standard LAMP procedure with agarose gel electrophoresis, described in the literature by Arunrut et al. [
29]. The sensitivity and repeatability test confirmed that the proposed LAMP–LOC platform can provide similar sensitivity and reliability for early detection of EMS at 10
2 copies as the other standard LAMP laboratory tests mentioned.
5. Conclusions
The LAMP–LOC platform was designed, fabricated with suitable and affordable materials, and tested with the aim of confirming the sensitivity and reliability of EMS detection. PTH-PCB was selected as a suitable electrode layer for the proposed LOC platform. The appropriate electrical control signal appeared to be AC at 700 Vrms and a frequency of 1 kHz in order to ensure the perfect manipulation of droplets on the proposed LAMP–LOC platform. A functional test was also carried out, which showed that the LAMP–LOC platform had a similar sensitivity (measured in terms of LOD) to the other LAMP laboratory tests described in this study. The LAMP–LOC platform can detect Vibrio parahaemolyticus (VP) infection at an EMS DNA plasmid concentration as low as 102 copies.
The proposed LAMP–LOC platform is also capable of the simultaneous detection of multiple diseases, which provides an advantage over the other conventional LAMP laboratory testing procedures and is crucial for field test applications. For example, in the case of shrimp pond infections, four different LAMP–XO premixes for the detection of EMS/AHPND disease, white spot disease (WSD), hepatopancreatic microsporidiosis disease (HPM) and hypodermal/hematopoietic necrosis virus infection (IHHNV), which are common severe diseases in shrimps [
36], can be applied to the four mixing stations, thus allowing the LAMP–LOC platform to diagnose four different types of shrimp diseases simultaneously using just one drop of a DNA sample.
In terms of the cost benefits, conventional PCR, qPCR and LAMP laboratory tests require thermal cyclers to enable thermal cycling amplification processes, while the simple heating box of the proposed LAMP–LOC platform can carry out similar tasks and eliminates the need for a thermal cycler costing USD 5000, instead utilizing a LAMP–LOC platform testing set that costs only USD 1700, as shown in
Figure 13. Overcoming further challenges with regard to replacing the function generator and power amplifier with a signal-generating circuit board would significantly reduce the cost of the LAMP–LOC testing platform to within USD 350.