SYBR-Green-Based Quantitative Real-Time PCR for Discriminating Between Closely Related Angiostrongylus Cantonensis and A. Malaysisensis

Background: Angiostrongylus cantonensis is a well-known pathogen causing human angiostrongyliasis eosinophilic meningitis. Humans, as accidental hosts, are infected by eating undercooked snails containing third-stage larvae. A. malaysiensis is closely related to A. cantonensis and has been described as a potential human pathogen. Recently, the two species have been reported to have overlapping distributions in the same endemic area, particularly in the Indochina region. Because of their similar morphological characteristics, misidentication often occurs, particularly of the third-stage larva in the snail intermediate host. Methods: We designed species-specic primers to mitochondrial cytochrome b, which was used as a genetic marker. SYBR-green quantitative real-time PCR (qPCR) was employed to quantitatively detect and identify the third-stage larvae and tissue debris in the cerebrospinal uid (CSF) of a patient, and to quantify third-stage larvae in the snail Achatina fulica collected from the eld. Results: The newly designed primers were highly specic and sensitive, even when using conventional PCR. SYBR green qPCR quantitatively detected around 10 −4 ng of genomic DNA from one larva and facilitated the specic detection and identication of parasitic genetic material from the CSF of a patient with angiostrongyliasis. The method also estimated the number of larvae in A. fulica and revealed that the primary source of Angiostrongylus infection in the King Rama IX public park study area was A. malaysiensis; although, the two Angiostrongylus species each infected 10% of the snails. Conclusions: Our SYBR green qPCR method is a useful and inexpensive technique for parasite identication and has sucient sensitivity and specicity to detect a single larva and simultaneously discriminate between A. cantonensis and A. malaysiensis. The number of larvae infecting or co-infecting the snail intermediate host can also be estimated. In future research, this qPCR method could be employed in a molecular survey of A. cantonensis and A. malaysiensis occurrence within intermediate and denitive hosts. The technique should also be applied in a study analyzing CSF specimens from patients The SYBR-green qPCR reactions were performed separately for each species using the species-specic primers. The qPCR was performed following the protocol of the Luna® Universal qPCR master mix (New England Biolabs, MA, USA). The reactions contained 10 µl of the master mix, 10 µM of each pair of species-specic primers, and 1 µl of gDNA, and RNAase-free water was added to a nal volume of 20 µl. The reaction was performed on a CFX96 TouchTM Real-Time PCR machine (Bio-Rad Laboratories, CA, USA) with the standard PCR conditions. The real-time PCR thermal cycle included an initial denaturation at 95°C for 60 s, followed by 45 cycles of denaturation at 95°C for 15 s, and an extension at 60°C for 30 s. A nal melting analysis program was applied at 60°C to 95°C and the duration of each cycle was 5 s with a 0.5°C increment per cycle. The SYBR-green qPCR described above was used for standard curve preparation, specicity and sensitivity assays, reproducibility assay, and implementation of the developed method to detect the third-stage larvae of Angiostrongylus in A. fulica. Serial qPCR.

in human samples and the discrimination of species and subspecies of Salmonella. The technique was also employed for the detection of Opisthorchis viverrini and Haplorchis taichui from human stool samples [28][29][30].
In this study, therefore, we designed species-speci c primers to Cytb partial gene sequences, which have su ciently high genetic variation between species to prevent cross-ampli cation [17,24]. We aimed to develop a highly sensitive and speci c method capable of quantitively detecting the third-stage larvae of A. cantonensis and A. malaysiensis in their intermediate host, Achatina fulica, and to determine the e cacy of the method for the detection of Angiostrongylus genomic material within the CSF of infected patients.

Specimens used
Reference specimens for evaluating sensitivity and speci city Third-stage larvae of A. cantonensis, adult worms of A. cantonensis and A. malaysiensis, CSF of patients with neurocysticercosis, gnathostomiasis, and angiostrongyliasis, and negative CSF of angiostrongyliasis were obtained from archived research specimen stock kept in the Department of Helminthology, Mahidol University. The selected CSF was from patients with diseases related to eosinophilic meningitis. The larvae and adult worms of Angiostrongylus were preserved in 70% ethanol at − 20°C, while the CSF specimens were kept at − 80°C. The specimens were used to test the sensitivity and speci city of the species-speci c primers designed for the SYBR green qPCR.
Experimental specimens to determining the validity of SYBR green qPCR Third-stage larvae of Angiostrongylus were collected from 48 A. fulica collected at King Rama IX Public Park in Bangkok, Thailand (geographical coordinates 13.68N, 100.65E). The snails were transported to the laboratory of the Department of Helminthology within 1 hour. The snails were then euthanized at 0°C for 10 min. Then, the shell of each snail was removed with a meat grinder. The foot and mantel parts of the snail were dissected and individually incubated with a digestion solution (1% HCl and 1% Pepsin) at 37°C for 1 hour. The digestion method used for the snails followed the protocol of Vitta et al. [31]. The larvae collected were identi ed as Angiostrongylus according to the morphological criteria described by Ash [32]. The larvae from each positive snail were approximately counted and preserved in 70% ethanol at − 20°C until used for DNA extraction. The wild snail collection was performed with permission from the The individual adult A. cantonensis and A. malaysiensis of the described reference specimens were transferred into a 1.7-ml centrifuge tube and washed thoroughly with sterile distilled water. Total genomic DNA (gDNA) was extracted from each specimen using the Genomic DNA mini kit (Geneaid Biotech Ltd, Taipei, Taiwan) following the manufacturer's instructions. The gDNA was eluted from the column with 30 µl of PCR-grade sterile water and the concentration measured using a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scienti c, MA, USA). The gDNA was stored at 4 °C until use.

Design of species-speci c primers
Species-speci c primers were designed manually based on the Cytb gene sequences of A. cantonensis (GenBank accession numbers KC995211, KC995223, KC995262) and A. malaysiensis (GenBank accession numbers KX147380, KX147406, KX147442). The properties of the oligonucleotide primers, including GC content, amplicon size, melting temperature, and hairpin formation, were predicted by OligoCalc version 3.27 and Primer3 [33,34]. Information on the designed primers is shown in Table S1.
Optimization of PCR conditions using conventional PCR Optimization of the PCR conditions was performed prior to the main quantitative reaction using conventional PCR, and the sensitivity and speci city of the newly designed primers were evaluated. Afterward, the optimized conditions for the species-speci c primers of A. cantonensis and A. malaysiensis were applied in the SYB-green qPCR.

Sensitivity test
The gDNA of adult A. cantonensis and A. malaysiensis reference specimens were prepared by 10-fold serial dilution (between 10 − 5 and 1 ng/µl) for use as DNA templates. The 20 µl PCR mixture contained 1 × One PCR™ Plus mixture (GeneDireX Inc., Taoyuan, Taiwan) 10 µM of species-speci c primers and 1 µl of gDNA template. The PCR was performed with an initial denaturation at 95°C for 5 min, followed by 34 cycles of denaturation at 95°C for 30 s, gradient primer annealing between 55°C and 60°C for 30 s, extension at 72°C for 30 s, and nal elongation at 72°C for 5 min. The reaction mixtures were then held at 12°C until the PCR products were collected. The PCR was conducted on a T100™ thermocycler (Bio-Rad Laboratories, CA, USA). The PCR products were run on 2% agarose gel at 50 V for 1 hour to determine the size of the amplicons.

Speci city test
Ten-fold serial dilutions of the gDNA from the reference specimens A. cantonensis and A. malaysiensis were used to determine the speci city of the primers. The DNA templates were used in an experimental mixture of species using different ratios of gDNA of A. cantonensis and A. malaysiensis, as described in Table S2. This was used to evaluate the robustness of further SYBR-green qPCRs. DNA ampli cation for the speci city test was performed following the PCR conditions described for the sensitivity test. The PCR products were run on 2% agarose gel at 50 V for 1 hour and stained with SYBR™ Safe (Life Technologies, CA, USA) to determine the species-speci c band sizes of A. cantonensis and A. malaysiensis. The PCR amplicons were then sequenced by the Sanger method with the PCR primers (Macrogen Inc., Seoul, Korea).

SYBR-green Quantitative Real-time PCR
The SYBR-green qPCR reactions were performed separately for each species using the species-speci c primers. The qPCR was performed following the protocol of the Luna® Universal qPCR master mix (New England Biolabs, MA, USA). The reactions contained 10 µl of the master mix, 10 µM of each pair of species-speci c primers, and 1 µl of gDNA, and RNAase-free water was added to a nal volume of 20 µl.
The reaction was performed on a CFX96 TouchTM Real-Time PCR machine (Bio-Rad Laboratories, CA, USA) with the standard PCR conditions. The real-time PCR thermal cycle included an initial denaturation at 95°C for 60 s, followed by 45 cycles of denaturation at 95°C for 15 s, and an extension at 60°C for 30 s. A nal melting analysis program was applied at 60°C to 95°C and the duration of each cycle was 5 s with a 0.5°C increment per cycle. The SYBR-green qPCR described above was used for standard curve preparation, speci city and sensitivity assays, reproducibility assay, and implementation of the developed method to detect the third-stage larvae of Angiostrongylus in A. fulica.

Standard curve construction for SYBR-green qPCR
Ten-fold serial dilutions of A. cantonensis and A. malaysiensis adult worm gDNA were used to construct the SYBR-green qPCR standard curve; gDNA concentrations of 10 − 4 to 1 ng/µl were used as DNA templates for qPCR, and each DNA concentration was ampli ed three times. The precision of the standard curve and robustness of the qPCR were veri ed by considering the slope values, correlation coe cient, and qPCR e ciency of both A. cantonensis and A. malaysiensis.

Speci city assay
The species-speci c primers developed for A. cantonensis and A. malaysiensis were evaluated for speci city to the various gDNA templates described in Table 2 using the SYBR-green qPCR pro le described above. The qPCRs were conducted three times for each gDNA template. The speci city of the qPCR was also tested with the CSF of patients with gnathostomiasis, cysticercosis, and angiostrongyliasis. The PCR products were then run on 2% agarose gel at 50 V for 1 hour and stained with SYBR™ Safe (Life Technologies, CA, USA) to determine the species-speci c band sizes with the positive controls for each Angiostrongylus species. The puri ed DNA samples were sequenced by Macrogen (Seoul, South Korea), an external biotechnology company, using the Sanger sequencing method with the primers used for the PCR ampli cation. The obtained nucleotide sequences (query sequences) were con rmed as the speci c species target sequences by comparing with annotated sequences in the NCBI databases using the standard nucleotide BLAST [35].

Sensitivity assay
To test the sensitivity of the SYBR-green qPCR method, we used gDNA from groups of 1, 5, 10, 50, 100, and 200 third-stage A. cantonensis larvae. Before gDNA isolation, the cuticles of the larvae were broken by bead beating with 20 mg of 0.1 mm silica beads in 200 µl lysis buffer using Tissue Lyser LT at 50 Hz for 30 s (Qiagen, Hilden, Germany). Each larval sample was beaten three times and then left on ice to prevent DNA degradation. The gDNA from each group of larvae was then extracted using the Tissue Genomic DNA Mini kit (Geneaid Biotech Ltd, Taipei, Taiwan). Subsequently, each sample was ampli ed three times with the SYBR-green qPCR pro le described above. The threshold cycle (C t ) or the quantitation cycle (Cq) values obtained for each group were estimated with the standard curve for the qPCR to determine the amount of larval gDNA.

Reproducibility assay
The 10-fold serial dilution of the standard DNA, which was used to construct the standard curve, was used in the reproducibility assay. The intra-reproducibility assay was estimated by amplifying three replicates of each DNA concentration using the qPCR pro le described above. Variations in the intrareproducibility assays were assessed. The experiments were repeated on 2 different days to determine the inter-reproducibility. The mean, SD, and coe cient of variation (CV) were calculated separately using the Ct values to evaluate the reproducibility of the developed SYBR-green qPCR. The percentage of the CV of inter-and intra-reproducibility assays were calculated to determine the consistency of the assay manipulation.
Detection of third-stage larvae of A. cantonensis and A. malaysiensis from naturally infected A. fulica The developed SYBR-green qPCR was implemented to detect and identify the third-stage larvae of Angiostrongylus collected from A. fulica. Before gDNA extraction, the larval cuticles were disrupted as described above. The larvae were then removed separately from each snail, and the DNA was extracted using the tissue genomic DNA mini kit (Geneaid, Taipei, Taiwan) according to the manufacturer's instructions. The extracted gDNA was then used as the template in the developed qPCR method. The standard curves were constructed to estimate the number of larvae and the ratio of A. cantonensis to A. malaysiensis in each A. fulica.

Results
Optimization of the newly designed species-speci c primers using conventional PCR Sensitivity and speci city tests with conventional PCR The PCR products from the 10-fold diluted gDNA of A. cantonensis and A. malaysiensis are shown in Figure S1. The different sizes of the species-speci c bands were used to discriminate between A. cantonensis (117 bp) and A. malaysiensis (141 bp). The ampli cations were successful for DNA concentrations of 10 − 4 to 1 ng/µl.
The mixed A. cantonensis and A. malaysiensis gDNA was used to evaluate the speci city of the newly designed species-speci c primers. Different ratios of gDNA from the two species, including 10 − 2 :10 − 1 ng, and vice versa, and 1:1 ng were ampli ed with conventional PCR using the above conditions. The sizes of the species-speci c PCR amplicons were used to determine the speci city of the designed primers (Fig.  S2). The DNA sequences of those PCR amplicons also con rmed the PCR speci city.
Standard curve for SYBR-green quantitative real-time PCR Sensitivity and speci city of SYBR green quantitative realtime PCR The sensitivities of the species-speci c primers used in SYBR-green qPCR were tested using the gDNA extracted from various A. cantonensis larvae group sizes from 1 to 200. The Cq values for the ampli ed larval gDNA were compared with those of the standard curve. Approximately 1 ng of gDNA was extracted from 100 larvae, while 10 − 4 ng of gDNA was extracted from 1 larva (Fig. 2A). The qPCR melting curve and the gel electrophoresis demonstrated the speci city of the primers to the A. cantonensis gDNA (Fig. 2B).
The speci city of the SYBR-green qPCR was determined using the gDNA template described in Table S2. Figure S3 shows the speci city of the A. cantonensis and A. malaysiensis-speci c primers. The speciesspeci c primers did not amplify the gDNA across different species. The high speci cities of the AC4_cytb_F and AC5_cytb_R primers for A. cantonensis were also demonstrated for the arti cially mixed A. cantonensis and A. malaysiensis gDNA, in both the 1:1 ratio and when there was less A. cantonensis than A. malaysiensis gDNA. These primers did not amplify Cytb from gnathostomiasis and neurocysticercosis samples or the heterogeneous sample containing 1 ng of A. malaysiensis gDNA. The speci city of the AC4_cytb_F and AC5_cytb_R primers was con rmed by the size of the qPCR amplicon (Fig. 3A). A speci city assay was then performed with AM3_cytb_F and AM4_cytb_R primers for A. malaysiensis. They also showed a speci city similar to the primers for A. cantonensis. The primers designed for A. malaysiensis did not amplify any Cytb from the CSF of patients, including those with angiostrongyliasis (Fig. 3B).

Intra-and inter-reproducibility assays of SRBR-green qPCR
Intra-and inter-reproducibility assays were performed similarly to the method used to construct the A.
cantonensis and A. malaysiensis gDNA standard curves. Serial concentrations of gDNA from 10 − 4 to 1 ng were ampli ed using SYBR-green qPCR. Three replicates of each DNA concentration were conducted for the intra-reproducibility assay, and the qPCRs were repeated over 3 days. The results showed there was consistent ampli cation between the replicated series of gDNA concentrations in the same assay (inter-reproducibility). The precision of the assay manipulation was considered to be the reproducibility of the qPCR reactions for A. cantonensis and A. malaysiensis, which is shown in Tables 1 and 2. The SYBR-green qPCR results showed that the species-speci c primers discriminated between A. cantonensis and A. malaysiensis third-stage larvae. All of the third-stage Angiostrongylus larvae collected from each snail were approximately counted, and DNA was extracted without excluding dead and weakened larvae. Larvae numbers in each snail were estimated as the degree of intensity, and the results are shown in Table 3. The qPCR results suggested there was no cross-ampli cation. The Angiostrongylus larvae detected from four infected snails were mainly A. malaysiensis, while two snails had coinfections of A. malaysiensis and A. cantonensis. There were fewer Angiostrongylus larvae counted from each snail than the numbers estimated by qPCR.

Discussion
In this study, we developed a simple, economical, highly sensitive, and speci c assay based on SYBRgreen qPCR for the detection, discrimination, and quanti cation of third-stage larvae of A. cantonensis and its closely related species A. malaysiensis from the intermediate host A. fulica. Recently, in some areas, and particularly areas in Indochina, there have been reports of overlap in the distributions of A. cantonensis and A. malaysiensis within the terrestrial snail host [23]. Although the gold standard for Angiostrongylus identi cation is the use of morphological characteristics, the characteristics of the thirdstage larvae of these two species are very similar [5,21,32] and misidenti cation often occurs.
We designed species-speci c primers to the mitochondrial Cytb gene of A. cantonensis and A. malaysiensis based on the genetic variation in the partial sequences. The two species can be differentiated by comparing the size of conventional PCR products for mitochondrial Cytb on gel electrophoresis (see Fig. S1). The high sensitivity of conventional PCR allows the ampli cation of 10 − 4 ng of gDNA. The newly designed primers showed high speci city without cross-species ampli cation when a heterogeneous gDNA sample containing a small amount of speci c DNA template was used (Fig.  S2). However, conventional PCR cannot quantify the number of Angiostrongylus larvae. Therefore, a qPCR method was developed that could detect a small amount of DNA (10 − 4 ng or less) from both Angiostrongylus species. We also established the standard curve and demonstrated the high e ciency of the qPCR reactions (see Fig. 1A and B). The % CV of reproducibility con rmed that the standard curve construction had low system variation in the manipulation assay. The number of larvae was then estimated using a standard curve constructed from 10-fold serial DNA dilutions. Based on the qPCR results, we postulated that DNA damage and loss during the larvae preservation and DNA extraction process might have resulted in the uctuation of the Cq value, particularly for a small amount of DNA. In contrast, there was no effect seen when more than 10 larvae were used ( Fig. 2A).
We also con rmed the high speci city and absence of cross-ampli cation of the method using heterogeneous gDNA in the SYBR-green qPCR reactions (Fig. 3). The developed method provides an alternative way of detecting the low amount of Angiostrongylus genetic material contained in CSF specimens. We also showed the potential of using SYBR-green qPCR for diagnosis using the CSF of the patients with an A. cantonensis infection, and there was no cross-reaction with other diseases that cause eosinophilic meningitis, such as cysticercosis and gnathostomiasis.
After proving the e cacy and consistency of the developed qPCR assay, we implemented it in the molecular discrimination of A. cantonensis and A. malaysiensis third-stage larvae from A. fulica collected in a suburban public park in Bangkok, Thailand. The number of larvae can be ascertained using the developed qPCR method and comparing the results to the standard curve (see Fig. 2A). We obtained relatively fewer numbers from the larvae count compared with the qPCR estimate because the larvae collected from the snails included those that were dead and dying. Therefore, when applying this method to a molecular survey of Angiostrongylus larvae, the report should indicate that the numbers obtained are approximate or only indicate the ratio between species. When small numbers of larvae are being studied and species discrimination without quanti cation is adequate, we suggest that conventional PCR is used with the species-speci c primers.
Although the developed method required the design of two species-speci c primers, which were tested using two separate DNA templates, the two sets of primers can be used together with the same qPCR conditions and are sensitive enough to detect a single target species. This method does not require the design of a separate probe, such as with the TaqMan qPCR technique, which can be complicated and expensive. Moreover, SYBR green qPCR appears to be more sensitive than the TaqMan probe-based qPCR [29,36]. The speci city of SYBR-green qPCR may also provide information regarding the amount of DNA ampli cation by using the melting curve analysis [37].
Previously, the TaqMan probe real-time PCR was developed using ribosomal internal transcribed spacer 1 as the genetic marker [38] for detecting third-stage A. cantonensis larvae in mollusks, and it was more sensitive than 18S rDNA-based conventional PCR [38,39]. However, the TaqMan qPCR was not designed to estimate larval numbers or the level of infection in the snails [38]. The technique of detecting the DNA of A. cantonensis was applied to the diagnoses of patients with eosinophilic meningitis [26]. Although the newly developed SYBR-green qPCR method, using the mitochondrial Cytb gene as a marker, has not yet been proven to be suitable for molecular diagnosis, it may provide an alternative method in the future.
The new technique has the potential to con rm the occurrence of A. malaysiensis infection in patients living in areas where the distributions of A. cantonensis and A. malaysiensis overlap.
Although the sensitivity assay for the developed qPCR was limited because of the lack of a veri ed source of A. malaysiensis larvae, such as a reference strain, an assessment of the amount of DNA from various numbers of A. cantonensis was used instead. The high precision of the technique was clear, as the mean Ct values for A. cantonensis and A. malaysiensis were similar between manipulation assays, even for low amounts of gDNA (Tables 3 and 4). This evidence can be used to infer that the approximate amount of gDNA from the various A. cantonensis larva numbers can be used to represent A. malaysiensis. The approximate number of A. malaysiensis larvae may be determined from the estimated number in the sensitivity test of A. cantonensis larvae ( Fig. 2A). The developed method has not yet been employed to detect larvae in A. fulica tissue quantitively. We suggest that larvae should be isolated from the snails before gDNA extraction to determine the accuracy of the larvae number estimated by the qPCR method.

Conclusions
The developed SYBR-green qPCR method is a useful and inexpensive alternative technique for molecular parasite detection. It has su ciently high sensitivity and speci city to simultaneously detect a single larva of Angiostrongylus and discriminate between larvae of A. cantonensis and A. malaysiensis. The developed qPCR method can also be used to estimate the number or ratio of larvae infecting or co-