Polymerase Chain Reaction Conditions Research Articles

A polymerase chain reaction (PCR) method to detect emerging multidrug-resistant Salmonella Infantis harbouring pESI plasmid in seafood.

Salmonella Infantis is an emerging multidrug-resistant pathogen worldwide due to the acquisition of a megaplasmid pESI (Plasmid of Emerging Salmonella Infantis). Reported initially from poultry, the distribution of pESI-harbouring S. Infantis in other food types, including seafood, is unknown. This study aimed to develop and optimize a PCR assay for detecting pESI plasmid in Salmonella and non-Salmonella Enterobacterales. A duplex PCR targeting the hilA gene and a pESI-associated gene of S. Infantis was designed, and the PCR conditions were optimized. The specificity and sensitivity of the assay were established using 119 Salmonella serovars and 51 non-Salmonella bacterial strains. All Salmonella isolates yielded hilA PCR product, while only pESI S. Infantis was positive for both hilA and pESI genes. No amplification product was obtained with the DNA of 51 non-Salmonella bacterial strains. The detection limit of the duplex PCR was 104 CFU/ml of pure culture of pESI S. Infantis. The sensitivity of detection in artificially spiked shrimp meat was 1 CFU/g after 6 h of enrichment in lactose broth, followed by 12 h of selective enrichment in the Rappaport-Vassiliadis medium. The duplex assay will help screen seafood for Salmonella in general and pESI S. Infantis in particular. Given its high sensitivity, the PCR will be a valuable tool for seafood quality assurance. This approach decreases the typical 3-6 days' identification time of Salmonella to less than 24 hours. S. Infantis carrying the highly transmissible megaplasmid (pESI) is a significant food safety concern. Given its rapid geographical spread and high antimicrobial resistant traits, it is necessary to have a molecular tool that detects pESI-harbouring Salmonella. This study successfully developed a duplex PCR assay that simultaneously detects Salmonella enterica and pESI S. Infantis. This molecular tool will help understand the distribution, sources, and spread of MDR plasmid in the food environment.

Sweet enhancers of polymerase chain reaction

Although faster and powerful, polymerase chain reaction (PCR) often failed to amplify targets efficiently. Numerous PCR enhancers have been used to increase the amplification efficiency of difficult DNA targets. However, there is no systematic comparison of their effects in normal and difficult PCR conditions. In this paper, we have selected nine different PCR enhancers that can promote the PCR amplification efficiency. We have compared their effect in Taq DNA polymerase thermostability, inhibitor resistance, and amplification of various DNA targets. Although the PCR enhancers more or less reduced the amplification efficiency of DNA fragments with moderate GC-content, they were able to improve the amplification efficiency and specificity of GC-rich fragments. Betaine outperformed the other enhancers in amplification of GC-rich DNA fragments, thermostabilizing Taq DNA polymerase, and inhibitor tolerance. Sucrose and trehalose showed similar effect in thermostabilizing Taq DNA polymerase and inhibitor tolerance, while they showed mildest inhibitory effect on normal PCR. For GC-rich region-containing long DNA fragment amplification, 1 M betaine, 0.5 M betaine + 0.2 M sucrose, or 1 M betaine + 0.1 M sucrose can be used to effectively promote the amplification, while keep their negative effect in amplification of normal fragment to a minimal level.

Comparative analysis of reverse-transcription-polymerase chain reaction for Aichivirus detection.

Aichivirus-A (AiV-A), a member of the Kobuvirus genus of the family Picornaviridae, was first reported in stool samples of patients with non-bacterial gastroenteritis in Aichi Prefecture, Japan, in 1989. AiV has been reported from in various aquatic environments, such as surface water and sewage, can be transmitted via the fecal-oral route through contaminated water. As AiV is known to acute gastroenteritis worldwide, developing methods for AiV detection from contaminated environments and food is required. In the present study, we established an effective polymerase chain reaction (PCR) method to detect AiV. Various real-time reverse transcription (RT)-PCR and conventional PCR methods for AiV detection were compared, and the limit of detection was confirmed by comparing the sensitivity at varied primer concentrations and PCR conditions. The final detection limits were 102 copy/μL in conventional PCR, and 101 copy/μL in the real-time RT-PCR. The optimized method used in this study might aid in detecting AiV contamination.

Mutagenic primer-based novel multiplex PCR-RFLP technique to genotype BECN1 SNPs rs10512488 and rs11552192.

Single Nucleotide Polymorphisms (SNPs) in candidate autophagy gene BECN1 could influence its functions thereby autophagy process. BECN1 noncoding SNPs were found to be significantly associated with neurodegenerative disease and type 2 diabetes mellitus. This study aimed to develop a simultaneous genotyping technique for two BECN1 SNPs (rs10512488 and rs11552192). A mutagenic primer-based approach was used to introduce a NdeI restriction site to genotype rs10512488 by Artificial-Restriction Fragment Length Polymorphism (A-RFLP) along with rs11552192 by Polymerase Chain Reaction (PCR)-RFLP. Multiplexing PCR and restriction digestion reactions were set up for simultaneous genotyping of both SNPs in 100 healthy individuals. Genotypic and allele frequencies were manually calculated, and the Hardy-Weinberg Equilibrium was assessed using the chi-square test. We successfully developed PCR and RFLP conditions for the amplification and restriction digestion of both SNPs within the same tube for genotyping. The results of genotyping by newly developed multiplexing PCR-RFLP technique were concordant with the genotypes obtained by Sanger sequencing of samples. Allelic frequencies of rs10512488 obtained were 0.15 (A) and 0.85 (G), whereas allelic frequencies of rs11552192 were 0.16 (T) and 0.84 (A). The newly developed technique is rapid, cost-effective and time-saving for large-scale applications compared to sequencing methods and would play an important role in low-income settings. For the first time, allelic frequencies of rs10512488 and rs11552192 were reported among the North Indian population.

Simultaneous Identification of Water Buffalo, Cattle, and Yak Meat and Horns by Multiplex Polymerase Chain Reaction (PCR)

Beef is an important source of protein for human consumption globally; in addition to beef, water buffalo and yak meat are widely consumed in Asia. Additionally, water buffalo horns are an integral part of traditional Chinese medicine. Adulteration of water buffalo horn with cattle and yak horn for economic gain has become a prevalent and pressing concern. This study used multiplex polymerase chain reaction (PCR) for the simultaneous identification of meat and horns from water buffalo, cattle, and yaks within a single PCR test. Species-specific primers were designed for multiplex PCR based on a comprehensive comparison of mitochondrial gene sequences with those of commonly edible and horned animals. Multiplex PCR conditions were optimized by considering the concentration of components and the annealing temperatures for each set of species-specific primer pairs. A total of 220 bp amplicons were obtained for water buffalo, 269 bp for cattle, and 346 bp for yak. Validation measures were used to eliminate interference from other animal sources. Simultaneous identification of the three species in a single assay presents substantial advantages over conventional species-specific PCR methods.

Optimization of RT-PCR methods for enterovirus detection in groundwater

Enteroviruses (EVs), which belong to the Picornaviridae family, infect individuals asymptomatically or cause mild symptoms (fever, runny nose, cough, skin rash, sneezing, mouth blister). Severe cases can cause various diseases, such as acute hemorrhagic conjunctivitis, aseptic meningitis, or myocarditis, especially in infants. These viruses can be transmitted via the fecal-oral route via contaminated water. In this study, we established a polymerase chain reaction (PCR) method for detecting EVs in water sample using Coxsackievirus B5 (CV-B5) and Echovirus 30 (E-30), which belong to species B of the four species of EVs (EV-A to D). Several methods have been investigated and compared for the detection of EVs, including real-time reverse transcription (RT) polymerase chain reaction and conventional RT-PCR. The most sensitive primer sets were selected, and the PCR conditions were modified to increase sensitivity. We also quantified the detection limits of real-time and conventional RT-PCR. The detection limits of conventional RT-PCR were detected in 105–106 copy/mL for CV-B5 and 106–107 copy/mL for E-30, respectively. This optimized method for detecting EVs is expected to contribute substantially to the investigation of EV outbreaks in water samples.

Real-Time PCR-Based Screening for Homozygous SMN2 Deletion Using Residual Dried Blood Spots.

The survival motor neuron 2 (SMN2) gene is a recognized modifier gene of spinal muscular atrophy (SMA). However, our knowledge about the role of SMN2-other than its modification of SMA phenotypes-is very limited. Discussions regarding the relationship between homozygous SMN2 deletion and motor neuron diseases, including amyotrophic lateral sclerosis, have been mainly based on retrospective epidemiological studies of the diseases, and the precise relationship remains inconclusive. In the present study, we first estimated that the frequency of homozygous SMN2 deletion was ~1 in 20 in Japan. We then established a real-time polymerase chain reaction (PCR)-based screening method using residual dried blood spots to identify infants with homozygous SMN2 deletion. This method can be applied to a future prospective cohort study to clarify the relationship between homozygous SMN2 deletion and motor neuron diseases. In our real-time PCR experiment, both PCR (low annealing temperatures) and blood (high hematocrit values and low white blood cell counts) conditions were associated with incorrect results (i.e., false negatives and positives). Together, our findings not only help to elucidate the role of SMN2, but also aid in our understanding of the pitfalls of current SMA newborn screening programs for detecting homozygous SMN1 deletions.

An ARMS-Multiplex PCR Targeting SARS-CoV-2 Omicron Sub-Variants.

As of November 2021, the SARS-CoV-2 Omicron variant had made its appearance, gradually replacing the predominant Delta variant. Since its emergence, the Omicron variant has been continuously evolving through more than 500 strains, most of which belong to five sub-variants known as BA.1, BA.2, BA.3, BA.4, and BA.5. The aim of this study was to develop a multiplex polymerase chain reaction (PCR) that will be able to distinguish the basic sub-variants of Omicron in a rapid and specific way. Full genome sequences of Omicron strains with high frequency and wide geographical distribution were retrieved by the NCBI Virus and ENA databases. These sequences were compared to each other in order to locate single nucleotide polymorphisms common to all strains of the same sub-variant. These polymorphisms should also be capable of distinguishing Omicron sub-variants not only from each other but from previously circulating variants of SARS-CoV-2 as well. Thus, specific primers targeting characteristic polymorphisms of the four Omicron main branches BA.1, BA.2, BA.4, and BA.5 were designed according to the principles of the amplification refractory mutation system (ARMS) and with the ability to react under multiplex PCR conditions. According to our results, the ARMS-multiplex PCR could successfully distinguish all Omicron sub-variants that carry the corresponding mutations.

Development of microsatellite markers for the invasive mosquito Aedes koreicus

BackgroundAedes koreicus is a mosquito species native to East Asia which has recently invaded several countries in Europe. In Italy, this mosquito was first detected in the North-East in 2011 and is now widely distributed in the entire northern part of the country. The development of specific genetic markers, such as microsatellites, is necessary to uncover the dispersal routes of this mosquito from its native areas and, eventually, to plan future control interventions.MethodsAvailable raw sequences of genomic DNA of Ae. koreicus were screened in silico using BLASTn to identify possible microsatellite-containing sequences. Specific primer pairs were then designed, and their efficiency was determined through polymerase chain reaction (PCR) on 32 individuals of Ae. koreicus collected in Italy. PCR conditions were optimised in three multiplex reactions. Genotyping of individual mosquitoes was performed on both single and multiplex PCR reactions. Finally, analysis of intra-population variation was performed to assess the level of polymorphism of the markers.ResultsMosquito genotyping provided consistent results in both single and multiplex reactions. Out of the 31 microsatellite markers identified in the Ae. koreicus genome raw sequences, 11 were polymorphic in the examined mosquito samples.ConclusionsThe results show that the 11 microsatellite markers developed here hold potential for investigating the genetic structure of Ae. koreicus populations. These markers could thus represent a novel and useful tool to infer the routes of invasion of this mosquito species into Europe and other non-native areas.Graphical

Clinical application of next generation sequencing-based haplotype linkage analysis in the preimplantation genetic testing for germline mosaicisms

BackgroundPreimplantation genetic testing (PGT) for monogenic disorders (PGT-M) for germline mosaicism was previously highly dependent on polymerase chain reaction (PCR)-based directed mutation detection combined with linkage analysis of short tandem repeats (STRs). However, the number of STRs is usually limited. In addition, designing suitable probes and optimizing the reaction conditions for multiplex PCR are time-consuming and laborious. Here, we evaluated the effectiveness of next generation sequencing (NGS)-based haplotype linkage analysis in PGT of germline mosaicism.MethodsPGT-M with NGS-based haplotype linkage analysis was performed for two families with maternal germline mosaicism for an X-linked Duchenne muscular dystrophy (DMD) mutation (del exon 45–50) or an autosomal TSC1 mutation (c.2074C > T). Trophectoderm biopsy and multiple displacement amplification (MDA) were performed for a total of nine blastocysts. NGS and Sanger sequencing were performed in genomic DNA of family members and embryonic MDA products to detect DMD deletion and TSC1 mutation, respectively. Single nucleotide polymorphism (SNP) sites closely linked to pathogenic mutations were detected with NGS and served in haplotype linkage analysis. NGS-based aneuploidy screening was performed for all embryos to reduce the risk of pregnancy loss.ResultsAll nine blastocytes showed conclusive PGT results. Each family underwent one or two frozen-thawed embryo transfer cycles to obtain a clinical pregnancy, and the prenatal diagnosis showed that the fetus was genotypically normal and euploid for both families.ConclusionsNGS-SNP could effectively realize PGT for germline mosaicism. Compared with PCR-based methods, the NGS-SNP method with increased polymorphic informative markers can achieve a greater diagnostic accuracy. Further studies are warranted to verify the effectiveness of NGS-based PGT of germline mosaicism cases in the absence of surviving offsprings.

A Multiplex PCR Method for Simultaneous Detection of Infectious Laryngotracheitis Virus and Ornithobacterium rhinotracheale

To date, many fluorescence- and gel-based multiplex polymerase chain reaction (PCR) assays have been developed for the simultaneous detection of multiple infectious agents of respiratory disease in poultry. However, PCR assays are not available for other important emerging respiratory bacteria, such as Ornithobacterium rhinotracheale (ORT). We aimed to fill this gap by establishing a new duplex PCR method for the simultaneous detection of infectious laryngotracheitis virus (ILTV) and ORT. Multiplex primer design software was used to select the compatible multiplex primer pairs. It was determined that an annealing temperature of 65 °C and an initial concentration of 2.5 pmol/µL for each primer set were the most suitable conditions for multiplex PCR. The assay was confirmed to be specific, as it only detected the target pathogens, even in the presence of six non-target agents. The limit of detection was up to 103 copies/µL of template DNA for both ILTV and ORT. In the screening of 304 field samples, 23, 88, and 44 were positive for both ILTV and ORT, solely for ILTV, and solely ORT, respectively.

Real Time PCR-based diagnosis of human visceral leishmaniasis using urine samples.

Diagnosis of visceral leishmaniasis (VL) through the detection of its causative agents namely Leishmania donovani and L. infantum is traditionally based on immunochromatographic tests, microscopy of bone marrow, spleen aspirates, liver or lymph node and differential diagnosis. While the first process has low specificity, the later one carries the risk of fatal hemorrhage. Over the last decade, multiple Polymerase Chain Reaction (PCR) based diagnosis has been developed using blood and urine sample with a varying degree of sensitivity and specificity, an issue worth improving for precision diagnosis. Earlier, we reported a PCR-based diagnosis of L. donovani in peripheral blood using a novel set of PCR primers with absolute specificity. Using the same set of primers and PCR conditions, here we describe diagnosis of L. donovani from urine, for a non-invasive, rapid and safe diagnosis. Diagnosis of VL was carried out using urine samples collected from clinically diagnosed VL patients (n = 23) of Bangladesh in Real Time PCR. Test results were validated by comparing blood samples from the same set of patients. Sensitivity and specificity of this diagnosis was analyzed using retrospective bone marrow samples, collected earlier from confirmed VL patients (n = 19). The method showed 100% sensitivity in detecting L. donovani in urine and corresponding blood and retrospective bone marrow samples, as well as 100% specificity in control groups. A Real Time PCR-based molecular detection system using urine sample is hereafter presented what could be a, non-invasive approach for VL detection with precision and perfection.

Early Evidence on Genetic Polymorphisms in Conferring A "Two-Hit" Propensity to Renal Injury in Asian Indian Children.

Congenital anomalies of the kidney and urinary tract (CAKUT) are a common cause of end-stage renal disease in children. While certain nephrogenic genes have been incriminated in these malformations, data to identify the frequency of gene polymorphisms in Asian Indian children with CAKUT are scarce. This study was done to identify the effect of polymorphisms in paired-box gene 2 (PAX2), bone morphogenetic protein (BMP)-4, angiotensin-converting enzyme (ACE), and angiotensin II receptor Type 2 (AGTR2) nephrogenic genes on the development of CAKUT. In this prospective cohort study, 158 children <12 years old (86 cases with CAKUT and 72 age-matched controls) were analyzed. DNA from both sets was extracted from peripheral blood using the Keygen DNA extraction kit, and single-nucleotide gene polymorphisms (SNPs) in PAX2, BMP-4, ACE, and AGTR2 nephrogenic genes were detected by polymerase chain reaction (PCR) using previously published primers and PCR conditions. The presence of A allele SNP for AGTR2 gene at rs3736556 was found to be significantly correlated with the development of ureteropelvic junction obstruction and vesicoureteral reflux (VUR) with the TT allelic genotype having a lower incidence of pelviureteric junction obstruction (odds ratio [OR] 0.18 [95% confidence interval [CI], 0.06-0.55], P = 0.01) and VUR (OR 0.31 [95% CI, 0.11-0.91], P = 0.03). Furthermore, on substratification of the patients with the presence of the A allele of AGTR2, 24 out of 27 patients with scarring were found to harbor the D allele of the ACE gene, thus predisposing them to further renal damage. This study points to early evidence in the implication of nephrogenic genes in development as well as predisposition to renal injury in Asian Indian patients with CAKUT.

Detection of Mycobacterium tuberculosis in clinical sputum by a unique gene in MTB strains called Conserved protein TB18.5 (TB18.5).

A rapid and accurate diagnosis increases the treatment effect and decreases the mortality of tuberculosis (TB) patients. The purpose of this study was to establish an accurate, unique, and rapid molecular diagnostic technique to screen Mycobacterium tuberculosis (MTB) from clinical sputum. A unique gene in MTB strains called conserved protein TB18.5 (TB18.5) was selected by bioinformatics analysis. Two pairs of primers were designed to amplify TB18.5 using the nested polymerase chain reaction (PCR) or quantitative real‐time PCR. Nine pathogens and the MTB strain were used to determine the specificity of the TB18.5 gene. The sensitivity assay was performed after optimizing the PCR conditions. The correct fragment was amplified when a 10 copy number template was used. A total of 232 sputum samples were collected from TB patients (from 2019 to 2020) to evaluate the accuracy of the molecular method in this study. MTB was first detected using the BACTEC MGIT‐960 culture test and the Gene Xpert MTB/RIF assay. Totals of 195 (84.05%), 182 (78.45%), and 162 (69.83%) sputum samples were determined to be infected with MTB using nested PCR, the Gene Xpert MTB/RIF assay, and the BACTEC MGIT‐960 culture test, respectively. In summary, a rapid, unique, and sensitive molecular method was established to diagnose TB infection in clinical sputum samples.

Development of a duplex PCR assay for detecting Theileria luwenshuni and Anaplasma phagocytophilum in sheep and goats.

Coinfections with the tick-borne pathogens Theileria luwenshuni and Anaplasma phagocytophilum can cause significant economic losses in sheep and goat farming. The difficulty in detecting these two pathogens by microscopic examination warrants the development of a rapid detection test to discriminate them. In this study, a duplex polymerase chain reaction (PCR) assay was developed to simultaneously detect T. luwenshuni and A. phagocytophilum. Alignment of the sequences from related pathogens allowed us to design a primer pair targeting the 18S ribosomal RNA gene in T. luwenshuni and generate a target product of 962bp, whereas a previously reported species-specific primer (SSAP2f/SSAP2r) for A. phagocytophilum was used in the same reaction to generate a product of 641bp. Genomic DNA from T. luwenshuni and A. phagocytophilum was 10-fold serially diluted for testing PCR sensitivity. Under the optimal PCR conditions we established, the lower limit of detection of the assay was 29.13fg/μL for T. luwenshuni and 1.53fg/μL for A. phagocytophilum, and PCR primers used in this study were confirmed to be 100% species-specific using other hemoparasites previously identified by other methods. No significant difference was found between conventional and duplex PCR protocols used to detect the two species. Our study provides an effective, sensitive, specific, and accurate tool for the diagnosis and epidemiological surveillance of mixed infections of the two pathogens in sheep and goats.

ESTANDARIZACIÓN DE LA TÉCNICA DE PCR PARA LA DETECCIÓN DE LA SECUENCIA ITS1 DE NECATOR AMERICANUS (STILES, 1902) Y CLONACIÓN DEL PRODUCTO PARA SU USO COMO CONTROL

Hookworm is generally caused by Necator americanus (Stiles, 1902), causing digestive symptoms and anemia. The diagnosis by coprology can have low sensitivity, especially in light parasite loads. The Polymerase Chain Reaction (PCR) is a sensitive and specific technique, which must be adapted to laboratory conditions and positive controls are necessary. The objective of this work was the standardization of the PCR technique for the amplification of the ITS-1 sequence of N. americanus in stool samples and its cloning for its use as a control. Three DNA extraction protocols were standardized (Phenol/ chloroform, Saline Precipitation, and Chelex® 100 Resin). Optimal reagent concentrations (MgCl2, BSA, dNTP, primers, and Taq polymerase) were determined, as well as the hybridization temperature and number of cycles. The analytical sensitivity and specificity of the technique were determined. For cloning, the ITS-1 sequence amplified by PCR was purified and ligated with the vector pGEM-T-Easy. Competent E. coli XL1Blue MRF` cells were transformed with the ligation mixture (pGEM-T-easy-Na-ITS1), recombinant colonies were identified and plasmid DNA was extracted from them. The best DNA extraction protocol was phenol / chloroform, the optimal conditions for PCR were; 1.5 mM MgCl2, 0.5 mg/mL BSA, 100 µM dNTP, 0.6 µM primers, and 1 U Taq polymerase, 56 °C hybridization temperature and 40 cycles. The optimal amounts of reagents were less than the amounts used by other authors, allowing saving of reagents. 100% specificity and an analytical sensitivity of 10 pg of DNA (extracted from stool samples), and 10 fg of the plasmid (with the cloned sequence) were obtained, which worked very well as positive control.

Compilation of real‐time PCR conditions toward the standardization of environmental DNA methods

AbstractThe recent development of the environmental DNA (eDNA) technique has dramatically facilitated investigations of the distribution and abundance of macroorganisms in the natural environment. Various real‐time polymerase chain reaction (PCR)‐based methods have been proposed for the detection and quantification of eDNA by independent researchers or groups. Although several eDNA reviews have outlined the critical requirements for experimental processes and provided guidelines for best practice, clear standardization of eDNA protocols is lacking. Here, we collected the PCR conditions of previous eDNA studies using real‐time PCR (n = 190), that is, the volume of the DNA template, PCR cycles, number of PCR replicates and PCR inhibition tests. Then, we synthesized the current development of real‐time PCR techniques for eDNA studies. Our synthesis has identified the critical issues that need to be addressed to design standardized protocols for eDNA monitoring that will enable broader uptake of eDNA technology.

Towards Point of Care Diagnostics: Visual Detection of Meningitis Pathogens Directly from Cerebrospinal Fluid

AbstractRapid and cost‐efficient methods for diagnostics of infectious diseases can expedite prescription of adequate treatment, thus shortening the patients’ recovery and reducing adverse side effects. In this study, we developed a procedure for visual detection of bacterial pathogens Gram‐negative Neisseria meningitidis and Gram‐positive Streptococcus pneumoniae directly from human samples of cerebrospinal fluid (CSF). The assay uses hybridization probes that can efficiently recognize folded single‐stranded DNA analytes due to their split design. The assay includes the following stages: PCR amplification of N. meningitidis and S. pneumoniae targeted sequences; λ exonuclease treatment of the polymerase chain reaction (PCR) amplicons, and detection of the amplicons by hybridization probes with visual signal output. The assay requires 2.0 h with hands‐on time of about 40 min, and a regular PCR thermal cycler. It detects down to 10 bacteria in 2 μL of cerebrospinal fluid. The assay works for both gram‐positive and gram‐negative bacteria and does not require optimization of PCR conditions. This development creates a basis for the point‐of‐care diagnostics of bacterial meningitis with visual signal output.

Identification of turtle shell and its preparations by PCR-DNA sequencing method

A Polymerase chain reaction (PCR) with DNA sequencing method has developed to identify the species of turtle shell specimens, skinned and bleached turtle shells, and its preparations. Two pairs of universal primer of mitochondrial gene, 12S rRNA (L:1373, H:1478) and cytochrome b (L:14181, H:15149), were applied to perform PCR and test for the optimal PCR conditions. The PCR products of about 165-bp and 376-bp in size were obtained from all 12 authentic turtle shells by primers of the 12S rRNA gene and the cytochrome b gene, respectively. Following DNA sequencing analysis of both bands without primers and GeneBank database search, DNA sequences of about 108-bp of 12S rRNA fragments were well differentiated among the 12 authentic specimens. However, DNA sequences of 307-bp of cytochrome b fragments were not completely distinguished for those authentic specimens. The PCR method with the primers of mitochondrial 12S-rRNA can also be successfully applied to the skinned and bleached turtle shell, and its powder preparation to identify their corresponding species. Thirty samples of skinned and bleached turtle shells were identified and categorized as three different species of Siebenrockiella crassicollis, Indotestudo elongata, and Cuora amboinensis, respectively. Furthermore, one extract and three powders of turtle shell preparations were identified as Siebenrockiella crassicollis, and one of turtle extract preparations was shown the cross-amplification with the other animal. Turtle jelly preparation, however, did unidentifiable. The results also indicated that the endangered species of turtle listed in CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) Appendix I and the prohibition of wild fauna announced by the Committee on Chinese Medicine and Pharmacy, Department of Health, Taiwan were not used as raw materials in the Chinese pharmaceutical manufactures.

Solid-Phase PCR-Amplified DNAzyme Activity for Real-Time FO-SPR Detection of the MCR-2 Gene.

The polymerase chain reaction (PCR) has been the gold standard molecular analysis technique for decades and has seen quite some evolution in terms of reaction components, methodology, and readout mechanisms. Nucleic acid enzymes (NAzymes) have been used to further exploit the applications of PCR, but so far the work was limited to the colorimetric G-quadruplex or fluorescent substrate cleaving NAzymes. In this study, a solid-phase, fiber optic surface plasmon resonance (FO-SPR) technique is presented as an alternative readout for PCR utilizing NAzymes. First, the surface cleavage activity of DNAzyme-extended amplicons (DNAzyme-amps) is established, followed by optimization of the PCR conditions, which are required for compatibility with the FO-SPR system. Next, by integrating the complement of a 10-23 DNAzyme into the primer pair, PCR-amplified DNAzyme-amps were generated, tested, and validated on qPCR for the detection of the antimicrobial resistance gene MCR-2. Once validated, this primer concept was developed as a one-step assay, driven by PCR-amplified DNAzymes, for FO-SPR-based sensitive and specific detection. Using gold nanoparticle labeled RNA-DNA hybrid strands as substrate for the DNAzyme, PCR-amplified DNAzyme-amps generated in the presence of MCR-2 gene were monitored in real-time, which resulted in an experimental limit of detection of 4 × 105 copy numbers or 6.6 fM. In addition, the DNAzyme-based FO-PCR assay was able to discriminate between the MCR-1 and MCR-2 genes, to further prove the specificity of this assay. Henceforth, this DNAzyme-based fiber optic PCR assay provides a universally applicable, real-time system for the detection of virtually any target NA, in a specific and sensitive manner.