Single Primer Isothermal Amplification Research Articles

Ultrasensitive detection of Salmonella typhi using a PAM-free Cas14a-based biosensor

The effectiveness of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas14a1, widely utilized for pathogenic microorganism detection, has been limited by the requirement of a protospacer adjacent motif (PAM) on the target DNA strands. To overcome this limitation, this study developed a Single Primer isothermal amplification integrated-Cas14a1 biosensor (SPCas) for detecting Salmonella typhi that does not rely on a PAM sequence. The SPCas biosensor utilizes a novel primer design featuring an RNA-DNA primer and a 3′-biotin-modified primer capable of binding to the same single-stranded DNA (ssDNA) in the presence of the target gene. The RNA-DNA primer undergoes amplification and is blocked at the biotin-modified end. Subsequently, strand replacement is initiated to generate ssDNA assisted by RNase H and Bst enzymes, which activate the trans-cleavage activity of Cas14a1 even in the absence of a PAM sequence. Leveraging both cyclic chain replacement reaction amplification and Cas14a1 trans-cleavage activity, the SPCas biosensor exhibits a remarkable diagnostic sensitivity of 5 CFU/mL. Additionally, in the assessment of 20 milk samples, the SPCas platform demonstrated 100% diagnostic accuracy, which is consistent with the gold standard qPCR. This platform introduces a novel approach for developing innovative CRISPR-Cas-dependent biosensors without a PAM sequence.

Single primer isothermal amplification for real-time and visual on-site detection of genetically modified crops by recognizing CP4-EPSPS gene

In response to the potential controversies on increasing circulation of genetically modified organisms (GMOs) in the global markets, the rapid and sensitive detection methods are essential for monitoring of GMOs to maintain consumers' rights. Herein, we first developed a rapid technique for the real-time and visual on-site detection of the genetically modified crops (specifically targeting CP4-EPSPS gene) by using single primer isothermal amplification (SPIA) combined with SYBR Green II. 19 GM crop events and 12 non-GM crop events were used to assess the specificity of the developed SPIA assay, which presented excellent specificity. The limit of detection of the developed SPIA method was as low as 4 copies/μL, which was better than that of the real-time PCR method (20 copies/μL). The SPIA method was further used to assess 30 of actual samples, and the experimental results revealed that 3 positive samples were found, which were in good agreement with those of the real-time PCR methods. Overall, the major advantages of the developed SPIA over real-time PCR are the lower LOD, simplicity, and inexpensiveness. The developed SPIA method provides a potential alternative for the real-time and visual on-site detection of GMOs, which also accelerates the development of highly sensitive nucleic acid testing techniques.

Novel Single Primer Isothermal Amplification Method for the Visual Detection of Vibrio parahaemolyticus

As a typical halophilic bacterium, Vibrio parahaemolyticus (V. parahaemolyticus) is regarded as one of the marine seafood-borne pathogens causing severe illnesses in humans and aquatic animals. Herein, a novel single primer isothermal amplification (SPIA) technique which was modified with SYBR Green II was successfully developed for the visual detection of V. parahaemolyticus. Interestingly, the visual superiority of SPIA can be achieved by fluorescence or precipitate to analyze the target pathogens in the seafood samples. The experimental results showed that all of V. parahaemolyticus strains were positive by using the novel SPIA assay, whereas the non-V. parahaemolyticus counterparts were negative. Furthermore, the limit of detection (LOD) of the SPIA assay was 5.7 CFU/mL in pure culture and 42 CFU/g in raw oysters by the visual fluorescence, respectively. Compared with the standard method, the developed SPIA approach achieved the satisfactory results with the characteristics of 100% sensitivity, 98.21% specificity, and 99.17% accuracy in the detection of 241 seafood samples. The developed SPIA method has been demonstrated as a rapid, specific, sensitive, and visual tool for the detection of V. parahaemolyticus, which could be adopted for routine surveillance of food-borne pathogens in the seafood samples.

‘Seq & Destroy’: the full genome sequencing of archived wild type and vaccine rinderpest virus isolates prior to their destruction

Rinderpest, a once much feared livestock disease, was declared eradicated in 2011, however virus-containing material is still held in laboratories worldwide. Prior to the destruction of our institute’s stocks, we determined the full genome sequence of the distinct samples of rinderpest virus (RPV) in our repository. This data would decipher the historical epidemiology of RPV and allow for recovery of the virus should the need arise. For each sample (n=123), sequencing libraries were prepared using either transposon-based fragmentation of cDNA (Nextera XT DNA Library Prep kit) or single primer isothermal amplification (Trio RNA-Seq kit) and sequenced on the Illumina MiSeq. Regions of low or no coverage were re-sequenced using a Sanger sequencing approach. Examination of the sequences of RPV isolates has shown that the African isolates form a single disparate clade, rather than two separate clades as was previously believed. We have also identified two groups of goat-passaged viruses which have acquired an extra 6 bases in the long untranslated region between the matrix and fusion protein coding sequences, and a group of African isolates where translation of the fusion protein begins from a non-standard start codon (AUA). In addition, the viruses that were force-passaged through alternate hosts such as rabbits or goats, appear to diverge from the clades that represent viruses which were maintained in the wild. Our unique set of sequence data will be invaluable for forensic epidemiology investigations in the event of an unforeseen outbreak and aid in the understanding of the evolution of related morbilliviruses.

Single primer isothermal amplification coupled with SYBR Green II: Real-time and rapid visual method for detection of Listeria monocytogenes in raw chicken

As a notorious foodborne pathogen worldwide, Listeria monocytogenes (L. monocytogenes) cause severe listeriosis with high mortality rate presenting a significant public health risk. Coupled with SYBR Green II, herein, a real-time and rapid visual single primer isothermal amplification (SPIA)method has been established for rapid detection of L. monocytogenes in raw chicken targeting the hlyA gene. The developed SPIA method enabled the resultscan be observed through the real-time amplification fluorescence curve, visual fluorescence or precipitate by naked eye. The method exhibited satisfied sensitivityof 1.4 × 101 CFU/mL by precipitate and 1.4 × 100 CFU/mL by fluorescence, respectively. With regard to the artificially inoculated raw chicken, the detection limits of SPIA were 1.4 × 101 CFU/g by precipitate and 1.4 × 100 CFU/g by fluorescence, respectively. In comparison with the conventional PCR method, the sensitivity of SPIA method was 10-fold or 100-fold better than that of PCR, while the detection limit was 10-fold or 100-fold lower than that of PCR. Compared with the ISO method, the relative sensitivity, specificity and accuracy of SPIA method were calculated as 100%, 97.47% and 97.65%, respectively. The results indicated that the developed SPIA method has the potential for rapid visual and on-site detection of L. monocytogenes in various food samples.

A Rapid and Visual Single Primer Isothermal Amplification-Based Method for the Detection of Staphylococcus aureus in Raw Pork Products

Staphylococcus aureus (S. aureus) is an important food-borne pathogen which poses a severe threat to public health worldwide. Rapid detection of S. aureus with high sensitivity is of particular importance for food safety. In this study, a novel single primer isothermal amplification (SPIA) method was established to detect S. aureus in food, targeting the accessory gene regulator (agr) gene with a DNA/RNA primer. The developed SPIA method has the advantages of visualization and avoiding tedious electrophoresis. In order to confirm the specificity of this method, 7 S. aureus strains and 26 non-S. aureus strains were detected with their pure cultures. The sensitivity and detection limit of S. aureus with artificially inoculated raw pork products by SPIA were evaluated through fluorescence and turbidity by naked eye and the amplification curve, which were 4.3 × 100 CFU/mL and 5.6 × 100 CFU/g, respectively. Compared with the conventional PCR method, the SPIA has 100-fold higher sensitivity and 100-fold lower detection limit. Therefore, the developed SPIA method is a potentially reliable tool for rapid and visual detection of S. aureus in food.

Single primer isothermal amplification (SPIA) combined with next generation sequencing provides complete bovine coronavirus genome coverage and higher sequence depth compared to sequence-independent single primer amplification (SISPA).

Coronaviruses are of major importance for both animal and human health. With the emergence of novel coronaviruses such as SARS and MERS, the need for fast genome characterisation is ever so important. Further, in order to understand the influence of quasispecies of these viruses in relation to biology, techniques for deep-sequence and full-length viral genome analysis are needed. In the present study, we compared the efficiency of two sequence-independent approaches [sequence-independent single primer amplification (SISPA) and single primer isothermal amplification (SPIA, represented by the Ovation kit)] coupled with high-throughput sequencing to generate the full-length genome of bovine coronavirus (BCoV) from a nasal swab. Both methods achieved high genome coverage (100% for SPIA and 99% for SISPA), however, there was a clear difference in the percentage of reads that mapped to BCoV. While approximately 45% of the Ovation reads mapped to BCoV (sequence depth of 169–284 944), only 0.07% of the SISPA reads (sequence depth of 0–249) mapped to the reference genome. Although BCoV was the focus of the study we also identified a bovine rhinitis B virus (BRBV) in the data sets. The trend for this virus was similar to that observed for BCoV regarding Ovation vs. SISPA, but with fewer sequences mapping to BRBV due to a lower amount of this virus. In summary, the SPIA approach used in this study produced coverage of the entire BCoV (high copy number) and BRBV (low copy number) and a high sequence/genome depth compared to SISPA. Although this is a limited study, the results indicate that the Ovation method could be a preferred approach for full genome sequencing if a low copy number of viral RNA is expected and if high sequence depth is desired.

Detection of <em>Cronobacter sakazakii</em> in Powdered Infant Formula using Real-time Fluorescence Single Primer Isothermal Amplification

Specific primers and blockers were designed based on Cronobacter sakazakii gene (16SrRNA) and Real-time Fluorescence Single Primer Isothermal Amplification (RF-SPIA) was established to detect C. sakazakii. This method identified that C. sakazakii genes were positive, whereas other non-C. sakazakii genes were negative. The sensitivity of this test for C. sakazakii pure culture reached 1.4×100CFU/mL and that for C. sakazakii genome reached 7.5×101fg/mL. The detection limit of RF-SPIA in milk powder artificially contaminated with C. sakazakii could reach 8.1×101CFU/mL. These results demonstrate that the RF-SPIA detection method for C. Sakazakii is highly sensitive, strong, specific and convenient.

A novel developed method based on single primer isothermal amplification for rapid detection of Alicyclobacillus acidoterrestris in apple juice

Alicyclobacillus acidoterrestris (A. acidoterrestris), as one of the most major spoilage-causing species within the Alicyclobacillus genus, can survive and multiply in the pasteurization process. Nowadays, A. acidoterrestris has become worldwide issue in the fruit juice industry due to its spore-forming and thermo-acidophilic features. A novel single primer isothermal amplification assay (SPIA) was developed for the rapid detection of A. acidoterrestris in apple juice. This assay was designed to target the 16S rRNA gene with a DNA/RNA chimeric primer. Detection of gene amplification was accomplished by amplification curve, turbidity and addition of single strand DNA binding dye SYBR Green II allowing visualization under ultra violet light. The specificity of the assay was confirmed using 7 strains of A. acidoterrestris and 30 strains of non-A. acidoterrestris. The SPIA was highly sensitive and the detection limit was as low as 4.8 CFU/mL. Furthermore, the apple juice inoculated with 61 CFU/mL of A. acidoterrestris could be detected as positive. The novel SPIA with visualization results was successfully applied for the detection of A. acidoterrestris and exhibited high specificity and sensitivity, making it a powerful tool for the detection of A. acidoterrestris in fruit juice industry and being conveniently applied in developing countries with limited resource.

Development of a quantitative fluorescence single primer isothermal amplification-based method for the detection of Salmonella

Food-borne disease caused by Salmonella has long been, and continues to be, an important global public health problem, necessitating rapid and accurate detection of Salmonella in food. Real time PCR is the most recently developed approach for Salmonella detection. Single primer isothermal amplification (SPIA), a novel gene amplification technique, has emerged as an attractive microbiological testing method. SPIA is performed under a constant temperature, eliminating the need for an expensive thermo-cycler. In addition, SPIA reactions can be accomplished in 30min, faster than real time PCR that usually takes over 2h. We developed a quantitative fluorescence SPIA-based method for the detection of Salmonella. Using Salmonella Typhimurium genomic DNA as template and a primer targeting Salmonella invA gene, we showed the detection limit of SPIA was 2.0×101fg DNA. Its successful amplification of different serotypic Salmonella genomic DNA but not non-Salmonella bacterial DNA demonstrated the specificity of SPIA. Furthermore, this method was validated with artificially contaminated beef. In conclusion, we showed high sensitivity and specificity of SPIA in the detection of Salmonella, comparable to real time PCR. In addition, SPIA is faster and more cost-effective (non-use of expensive cyclers), making it a potential alternative for field detection of Salmonella in resource-limited settings that are commonly encountered in developing countries.

A new molecular diagnosis method combined single primer isothermal amplification with rapid isothermal detection assay in detection of group B Streptococcus

Herein, a new molecular diagnosis assay for rapid detection of bacterial DNA was established, which combined single primer isothermal amplification (SPIA) with rapid isothermal detection assay (RIDA). Using this method, we could rapidly identify Group BStreptococcus DNA with high sensitivity and specificity, the detection limit of the new method was 102copy/mL. The whole assay was processed in one tube and the result was determined by naked eyes under ultraviolet radiation without cap-opening, meanwhile, as a linear amplification system, the products cannot be used as the amplification target, thus, the aerosol contamination was further decreased. This novel method shows excellent sensitivity, specificity and conveniences, which is easily operated in the elementary medical organizations and resource-limited areas.

Abstract 3073: Isothermal whole genome amplification of FFPE samples to detect copy number changes in the progression from colonic adenoma to carcinoma

Abstract Screening tumor genomes for somatic aberrations by aCGH is a commonly used high-resolution tool used both in research and clinical diagnostics. Unfortunately, the full utility of this technique is compromised with formalin fixed paraffin embedded (FFPE) tissue samples due to their inherently high degree of DNA degradation and the presence of DNA adducts acquired during the fixation and storage process. DNA degradation tends to worsen with storage time, making the analysis of archived samples even more challenging. We have developed a highly sensitive and robust whole genome amplification system for FFPE tumor samples that takes advantage of the proprietary Single Primer Isothermal Amplification (SPIA®) technology. The system uses 100 ng of routinely isolated FFPE DNA and produces 3-5 µg of DNA suitable for multiple downstream applications such as aCGH, enrichment strategies, and next generation sequencing (NGS). We evaluated the performance of the amplification system in the context of identifying aberrations by aCGH. Data will be presented that highlights the performance of the SPIA amplified material benchmarked against its unamplified counterpart along with the system's effectiveness on an array of sample qualities ranging from highly degraded FFPE DNA to highly intact FFPE DNA samples. Lastly, we employed the amplification system to examine the progression from adenoma to carcinoma using FFPE samples from human colon carcinomas within high-grade adenomas. Colonic epithelia gains numerous genetic alterations when degraded to dysplastic adenoma and then to cancer. While genetic alterations for each entity are well known, little data are available for combined lesions. Our results indicate that the SPIA amplified DNA performs equivalently to unamplified FFPE DNA in detecting copy number variations by aCGH, the system is suitable for a broad range of FFPE sample qualities, and is a flexible solution that allows for higher resolution analyses with NGS platforms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3073. doi:10.1158/1538-7445.AM2011-3073

Abstract 4826: Identification of low prevalence somatic mutations in heterogeneous tumor samples

Abstract High throughput sequencing enables the digital measurement of each allele in a DNA sample. This provides an ideal method to interrogate mutations present in heterogeneous samples such as solid tumors in which clonal selection or contamination with stroma can hinder the identification of important somatic mutations. We developed an ultra-deep targeted sequencing (UDT-Seq) assay to screen 46 cancer genes via microdroplet PCR (RainDance Technologies) and direct sequencing of the amplicons on the Illumina GA. This UDT-Seq library interrogates ∼86 kb of DNA located in cancer mutational hotspots (87% of all COSMIC mutations) and ∼29 kb located in exons sequenced in HapMap samples for the assay calibration and performance evaluation. We devised a statistical filtering of the mutations by using both experimental estimation and statistical modeling of the sequencing error rate. We measured the performance of our assay by processing 4 blends of 4 HapMap samples, interrogating 160 SNPs with known prevalence in each blend. The sensitivity and specificity of our method is >90% and >99% respectively for variants present at 5% prevalence or greater. We next interrogated 4 cancer samples (xenografts, 2 of which with matching primary samples) from 4 different tissues. We were able to detect low-prevalence somatic mutations in all samples of which some are well-known driver mutations. Interestingly, the mutational profile from primary to xenograft is conserved for one sample but different for another. Finally, we analyzed the robustness of the detection and prevalence measurement after performing whole-genome amplification on DNA extracted from fresh frozen and FFPE tissue using Single Primer Isothermal Amplification technology (SPIA® -NuGEN Technologies). Featuring a streamlined sample preparation to interrogate a large number of bases, this assay is well suited for clinical applications to study clonal selection in cancer progression or treatment with sub-optimal heterogeneous cancer samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4826. doi:10.1158/1538-7445.AM2011-4826

A review of the current isothermal amplification techniques: Applications, advantages and disadvantages

Polymerase chain reaction is the most widely used method for DNA amplification for the detection and identification of infectious diseases, genetic disorders and for other research purposes. However, it requires a thermocycling machine to separate the two DNA strands and then amplify the required fragment. Novel developments in molecular biology of DNA synthesis in vivo demonstrate the possibility of amplifying DNA in isothermal temperature without the need of a thermocycling apparatus. DNA polymerase replicates DNA with various accessory proteins. Therefore, with identification of these proteins, we are able to develop new in vitro isothermal DNA amplification methods by mimicking these in vivo mechanisms. There are several isothermal nucleic acid amplifications, such as transcription-mediated amplification or self-sustained sequence replication, nucleic acid sequence-based amplification, signal-mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single-primer isothermal amplification and circular helicase-dependent amplification. In this paper, we reviewed the nucleic acid amplification methods and their applications in molecular biology. We reviewed the best-known isothermal techniques for DNA/RNA amplification. The above information can be used for the application of valuable molecular diagnostic techniques for the detection of infectious agents in small-scale hospital laboratories in the field. With regards to the properties of isothermal DNA/RNA amplification techniques, the design and development of novel rapid molecular tests for application in field may be possible. ABSTRACT Address for correspondence:

Breast cancer patients stratification by microarray-based gene expression profiling from FFPET samples

e22041 Background: Microarrays have been used to monitor global genes expression and have aided the identification of novel biomarkers for patients stratification and drug response prediction . To date there has been limited application of microarray- based gene expression analysis to formalin fixed paraffin embedded tissues (FFPET). FFPE tissues are the most commonly available clinical samples with documented clinical information for retrospective clinical analysis. However, FFPET RNA has proven to be an obstacle for microarray analysis because of low yield and compromised RNA integrity. Methods: Using a novel RNA amplification method, Single Primer Isothermal Amplification (SPIA, NuGEN Inc, San Carlos, CA), we amplified FFPET RNA, hybridized amplified, and labeled cDNA onto Affymetrix HG U133plus2 GeneChips. Results: We found that SPIA amplification successfully overcomes the problems of poor quality of FFPET RNA, and produced informative biological data. Comparing the gene expression data from 5 different types of FFPET archival cancer samples (breast, lung, ovarian, colon, and melanoma), we demonstrated that gene expression signatures clearly distinguish the tissue of origin. Further, from an analysis of 91 FFPET samples comprised of ER+, HER2+, triple negative breast cancer patients, and normal breast tissue, we have identified a 103 gene signature that distinguishes the intrinsic sub-types of breast cancer. Finally, the accuracy of gene expression measured by microarray was verified by real time PCR quantitation of the ERBB2 gene, resulting in a significant correlation (R = 0.88). Conclusions: We have demonstrated the feasibility of global gene expression profiling using RNA extracted from FFPET and have shown that a gene expression signature can stratify patient samples into different subtypes of disease. This study paves the way to identify novel molecular biomarkers for disease stratification and therapy response from archival FFPET samples, leading to the goals of personalized medicine. No significant financial relationships to disclose.

Nucleic Acid Isothermal Amplification Technologies—A Review

Nucleic acid amplification technologies are used in the field of molecular biology and recombinant DNA technologies. These techniques are used as leading methods in detecting and analyzing a small quantity of nucleic acids. The polymerase chain reaction (PCR) is the most widely used method for DNA amplification for detection and identification of infectious diseases, genetic disorders and other research purposes. However, it requires a thermocycling machine to separate two DNA strands and then amplify the required fragment. Novel developments in molecular biology of DNA synthesis in vivo demonstrate the possibility of amplifying DNA in isothermal conditions without the need of a thermocycling apparatus. DNA polymerase replicates DNA with the aid of various accessory proteins. Recent identification of these proteins has enabled development of new in vitro isothermal DNA amplification methods, mimicking these in vivo mechanisms. There are several types of isothermal nucleic acid amplification methods such as transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification. In this article, we review these isothermal nucleic acid amplification technologies and their applications in molecular biological studies.