Carryover Contamination Research Articles

Label-free and sensitive detection of uracil-DNA glycosylase using exponential real-time rolling circle amplification

Schematic representation of an exponential rolling circle amplification-based uracil-DNA glycosylase activity-sensing platform.

An advanced uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) technique used in the sensitive and specific detection of Cryptosporidium parvum, Cryptosporidium hominis, and Cryptosporidium meleagridis in AIDS patients

The rapid and accurate detection of Cryptosporidium spp. is critically important for the prevention and timely treatment of cryptosporidiosis in AIDS patients (APs). This study was conducted to examine a UDG-LAMP technique for the first time to diagnose cryptosporidiosis in APs. After collecting demographic and clinical data, three stool samples were collected from the participants (120 volunteering APs). The microscopic examination of stained smears using the acid-fast method and the UDG-LAMP assay were performed for each sample. 10% of APs were infected with Cryptosporidium spp. The number of detected cryptosporidiosis cases using the acid-fast staining and UDG-LAMP methods were significantly different (P < 0.001). Diarrhea and weight loss were found to be significantly associated with cryptosporidiosis in patients (P < 0.05). The pretreatment of LAMP reagents with UDG successfully eliminated the likelihood of product re-amplification remaining from previous reactions. The UDG-LAMP technique could detect cryptosporidiosis in APs with high sensitivity and rapidity without carryover contamination.

Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

Expanded CAG/CTG repeats underlie the aetiology of 14 neurological and neuromuscular disorders. The size of the repeat tract determines in large part the severity of these disorders with longer tracts causing more severe phenotypes. Expanded CAG/CTG repeats are also unstable in somatic tissues, which is thought to modify disease progression. Routine molecular biology applications involving these repeats, including quantifying their instability, are plagued by low PCR yields. This leads to the need for setting up more PCRs of the same locus, thereby increasing the risk of carry-over contamination. Here we aimed to reduce this risk by pre-treating the samples with a Uracil N-Glycosylase (Ung) and using dUTP instead of dTTP in PCRs. We successfully applied this method to the PCR amplification of expanded CAG/CTG repeats, their sequencing, and their molecular cloning. In addition, we optimized the gold-standard method for measuring repeat instability, small-pool PCR (SP-PCR), such that it can be used together with Ung and dUTP-containing PCRs, without compromising data quality. We performed SP-PCR on myotonic-dystrophy-derived samples containing an expansion as large as 1000 repeats, demonstrating the applicability to clinically-relevant material. Thus, we expect the protocols herein to be applicable for molecular diagnostics of expanded repeat disorders.

Loop-mediated isothermal amplification using self-avoiding molecular recognition systems and antarctic thermal sensitive uracil-DNA-glycosylase for detection of nucleic acid with prevention of carryover contamination

Loop-mediated isothermal amplification (LAMP) is the most popular technique to amplify nucleic acid sequence without the use of temperature cycling. However, LAMP is often confounded by false-positive results, arising from interactions between (hetero-dimer) or within (self-dimerization) primers, off-target hybrids and carryover contaminants. Here, we devised a new LAMP technique that is self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme-assisted, termed AUDG-SAMRS-LAMP. Incorporating SAMRS components into 3′-ends of LAMP primers can improve assay's specificity, which completely prevents the non-specific amplification yielding from off-target hybrids and undesired interactions between or within primers. Adding AUDG into reaction mixtures can effectively eliminate the false-positive results arising from carryover contamination, thus the genuine positive reactions are generated from the amplification of target templates. Furthermore, AUDG-SAMRS-LAMP results are confirmed using a new analysis strategy, which is developed for detecting LAMP amplicons by lateral flow biosensor (LFB). Only a single labeled primer is required in the analysis system, thus the false positive results arising from hybridization (the labeled primer and probe, or between two labeled primers) are avoided. Hence, the SAMRS components, AUDG and LFB convert traditional LAMP from a technique suited for the research laboratory into one that has practical value in the field of diagnosis. Human Tuberculosis (TB) is caused by infection with members of Mycobacterium tuberculosis complex (MTC), which are detected by the AUDG-SAMRS-LAMP technique to demonstrate the availability of target analysis. The proof-of-concept method can be reconfigured to detect various nucleic acids by redesigning the specific primers.

A label-free technique for accurate detection of nucleic acid–based self-avoiding molecular recognition systems supplemented multiple cross-displacement amplification and nanoparticles based biosensor

Here, we devised a novel isothermal technique on the basis of standard multiple cross-displacement amplification (MCDA), which is assisted with self-avoiding molecular recognition system (SAMRS) components and antarctic thermal-sensitive uracil-DNA-glycosylase enzyme (AUDG), termed AUDG–SAMRS–MCDA. To enable product detection on the dipsticks, we firstly developed an analysis strategy, which did not require the labelled primers or probes, and thus, the analysis system avoids the false-positive results arising from undesired hybridization (between two labelled primers, or the labelled probe and primer). The SAMRS components are incorporated into MCDA primers for improve the assay’s specificity, which can prevent the false-positive results yielding from off-target hybrids, undesired interactions between (hetero-dimer) or within (self-dimerization) primers. Two additional components (AUDG enzyme and dUTP) were added into the reaction mixtures, which were used for removing the false-positive results generating from carryover contamination, and thus, the genuine positives results were produced from the amplification of target templates. For the demonstration, the label-free AUDG–SAMRS–MCDA technique was successfully applied to detect Pseudomonas aeruginosa from pure culture and blood samples. As a proof-of-concept technique, the label-free AUDG–SAMRS–MCDA method can be reconfigured to detect different target sequences by redesigning the specific primers.

Modified RS-LAMP assay and use of lateral flow devices for rapid detection of Leifsonia xyli subsp. xyli.

This study presents a modified loop-mediated isothermal amplification (LAMP) assay for the detection of Leifsonia xyli subsp. xyli. Modifications include incorporation of uracil nucleoside glycosylase to eliminate carry-over contamination and substitution of colorimetric detection for the use of lateral flow devices. LAMP master mix was preprepared and was stably stored up to 4months at -20°C. Sugarcane leaf sheaths worked well as a substitute to xylem sap as template, although the sensitivity was lower. These modifications allow the assay to be conducted without contamination concerns and reduction in set up time, making it ideal for near-field diagnosis.

Nanoparticles-based lateral flow biosensor coupled with multiple cross displacement amplification Plus for simultaneous detection of nucleic acid and prevention of carryover contamination

Abstract Isothermal amplification techniques, which avoid thermal cycling, are often confounded by non-specific amplification arising from off-target hybrids, interactions between (hetero-dimer) or within (self-dimerization) primers and carryover contaminants. Here, we developed a new method, termed multiple cross displacement amplification Plus (MCDA Plus), which uses self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme to improve the specificity of MCDA and prevent carryover contamination, respectively. In the presence of SAMRS components, the false positive results arising from off-target hybrids and undesired interactions between or within primers are completely eliminated, which increase the reproducibility of the assay. In the presence of AUDG, the false positive results generating from carryover contaminants are removed, thus the genuine positive results are obtained from the amplification of target sequences. Moreover, we also devised a new analysis strategy, which was intended for the detection of amplified products using lateral flow biosensor (LFB). The analysis system only required a single labeled primer, thus eliminated the false positive result yielding from hybridization (the labeled primer and probe, or between two labeled primers). Therefore, the SAMRS components, AUDG and LFB convert standard MCDA from an assay suited only for the research laboratory into one that has applicable value at points-of-care testing, in the field environments and in poor-resource settings. For demonstration purpose, Mycobacterium tuberculosis complex (MTC), which is the causes of human Tuberculosis (TB), are detected by the novel MCDA Plus technique to demonstrate the capability of target analysis. The performance of MCDA Plus assay in identifying MTC from pure culture and clinical samples is successfully evaluated. The proof-of-concept technique can be reconfigured to detect various nucleic acid sequences by redesigning the specific MCDA Plus primers.

Evaluation of improved IS6110 LAMP assay for diagnosis of pulmonary and extra pulmonary tuberculosis

In the present study, IS6110 loop mediated isothermal amplification (LAMP) assay was modified using dUTP-UNG (uracil-DNA N-glycosylase) strategy to prevent carryover contamination, and was evaluated using clinical specimens. The clinical specimens were collected from 236 suspected patients of pulmonary tuberculosis and 315 specimens of suspected patients of extra pulmonary tuberculosis. DNA was extracted from specimens and used as template for nucleic acid amplification. The results were evaluated with culture method as gold standard. Modified IS6110 LAMP assay showed high sensitivity (94.4%) and specificity (97.2%) in specimens collected from suspected pulmonary tuberculosis patients. Sensitivity was comparatively less (86.67%) in extra pulmonary specimens while specificity was 94.04%. In conclusion, IS6110 LAMP assay was modified to prevent carry over contamination and it was validated to be rapid, sensitive and specific method with prospective application in resource-limited settings.

A novel, multiplexed, probe-based quantitative PCR assay for the soybean root- and stem-rot pathogen, Phytophthora sojae, utilizes its transposable element.

Phytophthora root rot of soybean [Glycine max (L.) Merr.] is caused by the oomycete Phytophthora sojae (Kaufm. & Gerd.). P. sojae has a narrow host range, consisting primarily of soybean, and it is a serious pathogen worldwide. It exists in root and stem tissues as mycelium, wherein it can form oospores which subsequently germinate to release motile, infectious zoospores. Molecular assays detecting DNA of P. sojae are useful in disease diagnostics, and for determining the presence of the organism in host tissues, soils, and runoff or ponded water from potentially infested fields. Such assays as published have utilized ITS sequences from the nuclear ribosomal RNA genes in conventional PCR or dye-binding quantitative PCR (Q-PCR) but are not amenable to multiplexing, and some of these assays did not utilize control strategies for type I or type II errors. In this study, we describe primers and a bifunctional probe with specificity to a gypsy-like retroelement in the P. sojae genome to create a fluorogenic 5’-exonuclease linear hydrolysis assay, with a multiplexed internal control reaction detecting an exogenous target to validate negative calls, and with uracil-deglycosylase-mediated protection against carryover contamination. The assay specifically detected 13 different P. sojae isolates, and excluded 17 other Phytophthora species along with 20 non-Phytophthora fungal and oomycete species pathogenic on soybean. A diagnostic limit of detection of 34 fg total P. sojae DNA was observed in serial dilutions, equivalent to 0.3 genome, and a practical detection sensitivity of four zoospores per sample was achieved, despite losses during DNA extraction.

Salmonella detection in powdered dairy products using a novel molecular tool

In this study, we developed a rapid, specific, and sensitive loop-mediated isothermal amplification technique combined with a lateral flow dipstick (LAMP-LFD) method to detect Salmonella targeting the siiA gene in powdered infant formula (PIF). The specificity of the detection method (LAMP-LFD) approached 100% using 21 Salmonella and 31 non-Salmonella bacterial strains. This detection method exhibited high sensitivity limits for pure cultures at 3.7 cfu/mL and in PIF at 2.2 cfu/g without enrichment. To evaluate the applicability of the LAMP-LFD method, we detected 60 positive PIF samples and 20 negative PIF samples. The results showed that the method of LAMP-LFD had a high diagnostic specificity of 100% for detection of Salmonella in PIF. To reduce incidence of LAMP contamination, we applied propidium monoazide (PMA) to eliminate carryover contamination of LAMP. At the same time, we found that PMA does not affect observation of LFD for measurement of LAMP signal. The results verified that the method of LAMP-LFD targeting the siiA gene is rapid, accurate, and sensitive for Salmonella detection in PIF, and that PMA shows great potential to be widely used to eliminate the amplicon contamination risk generated by the highly sensitive LAMP reaction in the detection process.

Abstract P1-03-11: Analytical validation for the RT-qPCR based multiplex mRNA measurements of ER, PgR, HER2, and Ki67 from FFPE tumor tissue using the GeneXpert breast cancer stratifier assay

Abstract BACKGROUND: Accurate assessment of ER, PgR, HER2 and Ki67 status is crucial for breast cancer therapy and patient management. Immunohistochemistry (IHC) assays have been standard diagnostic tools but they are complex and time-consuming to perform and may not be readily available in decentralized laboratories, particularly in low-to-middle income countries. Molecular diagnostics can be a sensitive and accurate alternative to the traditional IHC, and the GeneXpert Breast Cancer Stratifier assay RUO (BC Strat), a single use cartridge-based assay performed on the broadly distributed GeneXpert® Instrument (GX) platform, streamlines a technically demanding RT-qPCR process to provide easy, robust, and reproducible ESR1, PGR, ERBB2, and MKi67 mRNA measurements from a 4 µm thick, formalin-fixed paraffin embedded (FFPE) breast tumor section in less than 2 hours. METHODS: Analytical validation of the BC Strat assay included studies of Linearity/Dynamic Range, Analytical Sensitivity (Minimum Assay Input), Specificity (Potential Interfering Substances), Carryover Contaminations, and Kit and Specimen Slide Stabilities. Both in-vitro RNA transcript (IVT) and/or clinical breast cancer tissues were used as sample input materials. Assay results for each analyte were generated from cycle threshold (Ct) values, and final positive/negative test results for each target were also reported as delta Ct values, where dCt = Ct [CYFIP1 Reference] – Ct [Target], using dCt cutoffs previously derived from a clinical sample cohort. RESULTS: The BC Strat assay demonstrated ≥3 log Linear Dynamic Range covering 5-7 logs sample input for all 4 Target dCts with R2≥0.95 independently. The assay currently requires minimal sample input equivalent to CYFIP1 Ct≤35 (Ct=33.5 ±1.5Ct SD) from 20 replicates of 5-level serial sample dilutions using two independent assay lot materials. It is acceptably robust against non-tumor tissues, DCIS, necrotic and/or hemorrhagic cells, lymphocytes, and genomic DNA contaminants. No carryover contamination from the same GeneXpert module was observed over 20 repeat tests during 9 consecutive days. Current real-time data supports assay stability at 5, 30, 37, 45 and 50°C for at least 3 months with minimal performance impact. Sectioned FFPE breast tumor tissues generated consistent dCt results when stored at 4°C and 30°C for up to 1 month before BC Strat assay testing. CONCLUSIONS: The analytical validations of the BC Strat assay demonstrate an easy and robust mRNA detection with high sensitivity, specificity, reproducibility, and stability in order to aid medical pathologists and clinicians to more rapidly and objectively determine ESR1, PGR, ERBB2, and MKi67 mRNA status in breast cancer. Although stability studies out to 37 months are ongoing, current data suggest the assay is stable for at least 3 months over a wide range of temperatures. The GeneXpert Breast Cancer Stratifier assay potentially offers a rapid, standardized, and cost-effective solution to streamlining complex molecular diagnostics available for use in local pathology laboratories worldwide. Citation Format: Chu VC, Wu N, Ho KE, Rizo A, Malek M, Weidler JM, Bates M, Wong W. Analytical validation for the RT-qPCR based multiplex mRNA measurements of ER, PgR, HER2, and Ki67 from FFPE tumor tissue using the GeneXpert breast cancer stratifier assay [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-11.

Real-time PCR strategy for the identification of Trypanosoma cruzi discrete typing units directly in chronically infected human blood

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a major public health problem in Latin America. This parasite has a complex population structure comprised by six or seven major evolutionary lineages (discrete typing units or DTUs) TcI-TcVI and TcBat, some of which have apparently resulted from ancient hybridization events. Because of the existence of significant biological differences between these lineages, strain characterization methods have been essential to study T. cruzi in its different vectors and hosts. However, available methods can be laborious and costly, limited in resolution or sensitivity. In this study, a new genotyping strategy by real-time PCR to identify each of the six DTUs in clinical blood samples have been developed and evaluated.Two nuclear (SL-IR and 18S rDNA) and two mitochondrial genes (COII and ND1) were selected to develop original primers. The method was evaluated with eight genomic DNA of T. cruzi populations belonging to the six DTUs, one genomic DNA of Trypanosoma rangeli, and 53 blood samples from individuals with chronic Chagas disease. The assays had an analytical sensitivity of 1–25fg of DNA per reaction tube depending on the DTU analyzed. The selectivity of trials with 20fg/μL of genomic DNA identified each DTU, excluding non-targets DTUs in every test. The method was able to characterize 67.9% of the chronically infected clinical samples with high detection of TcII followed by TcI.With the proposed original genotyping methodology, each DTU was established with high sensitivity after a single real-time PCR assay. This novel protocol reduces carryover contamination, enables detection of each DTU independently and in the future, the quantification of each DTU in clinical blood samples.

A novel method to control carryover contamination in isothermal nucleic acid amplification

We developed a novel method to control carryover contamination in loop-mediated isothermal amplification (LAMP) by primer engineering to carry recognition sites for a restriction endonuclease, providing a robust ability to eliminate carryover contaminants.

Molecular Characterization of Infectious Bronchitis Virus in Chicken

INFECTIOUS BRONCHITIS (IB) is a highly contagious ………disease of serious economic importance in the poultry industry worldwide. Diagnosis of Infectious Bronchitis Virus (IBV) infections in Egypt during 2013 - 2016 among poultry flocks. A total of 41 chicken flocks were examined (40 broiler flock and one breeder flock). Results revealed that 21 chicken flocks were found positive IBV by N gene conventional RT-PCR and real time RT-PCR. The samples were isolated and propagated in commercial embryonated chicken eggs (ECE) with result revealed that only one sample that showed curling and dwarfing of the embryo after the second passage while 10 samples were HA positive that suggest mixed infection of IBV with other viruses such as Newcastle Virus or Avian Influenza Virus. Furthermore, the allantoic fluids of the positive samples were subjected to specific S1 PCR to differentiate strains of IBV. The absence of positive reaction of S1gene conventional RT-PCR may confirm the involvements of other respiratory pathogens in chickens as Newcastle disease, avian influenza and mycoplasmosis that masked IBV infections. In conclusion, our results confirmed the detection of IBV in chicken flocks in Egypt which responsible for the respiratory problems in poultry farms and diagnosis with Real-time PCR because it is more rapid, sensitive, and reproducible, and the risk of carryover contamination is minimized compared to conventional PCR.

A flow-cytometry-based method for detecting simultaneously five allergens in a complex food matrix.

To avoid carry-over contamination with allergens, food manufacturers implement quality control strategies relying primarily on detection of allergenic proteins by ELISA. Although sensitive and specific, this method allowed detection of only one allergen per analysis and effective control policies were thus based on multiplying the number of tests done in order to cover the whole range of allergens. We present in this work an immunoassay for the simultaneous detection of milk, egg, peanut, mustard and crustaceans in cookies samples. The method was based on a combination of flow cytometry with competitive ELISA where microbeads were used as sorbent surface. The test was able to detect the presence of the five allergens with median inhibitory concentrations (IC50) ranging from 2.5 to 15mg/kg according to the allergen to be detected. The lowest concentrations of contaminants inducing a significant difference of signal between non-contaminated controls and test samples were 2mg/kg of peanut, 5mg/kg of crustaceans, 5mg/kg of milk, 5mg/kg of mustard and 10mg/kg of egg. Assay sensitivity was influenced by the concentration of primary antibodies added to the sample extract for the competition and by the concentration of allergenic proteins bound to the surface of the microbeads.

TriXY—Homogeneous genetic sexing of highly degraded forensic samples including hair shafts

Sexing of biological evidence is an important aspect in forensic investigations. A routinely used molecular-genetic approach to this endeavour is the amelogenin sex test, which is integrated in most commercially available polymerase chain reaction (PCR) kits for human identification. However, this assay is not entirely effective in respect to highly degraded DNA samples. This study presents a homogeneous PCR assay for robust sex diagnosis, especially for the analysis of severely fragmented DNA. The introduced triplex for the X and Y chromosome (TriXY) is based on real-time PCR amplification of short intergenic sequences (<50bp) on both gonosomes. Subsequent PCR product examination and molecular-genetic sex-assignment rely on high-resolution melting (HRM) curve analysis. TriXY was optimized using commercially available multi-donor human DNA preparations of either male or female origin and successfully evaluated on challenging samples, including 46 ancient DNA specimens from archaeological excavations and a total of 16 DNA samples extracted from different segments of eight hair shafts of male and female donors. Additionally, sensitivity and cross-species amplification were examined to further test the assay’s utility in forensic investigations. TriXY's closed-tube format avoids post-PCR sample manipulations and, therefore, distinctly reduces the risk of PCR product carry-over contamination and sample mix-up, while reducing labour and financial expenses at the same time. The method is sensitive down to the DNA content of approximately two diploid cells and has proven highly useful on severely fragmented and low quantity ancient DNA samples. Furthermore, it even allowed for sexing of proximal hair shafts with very good results. In summary, TriXY facilitates highly sensitive, rapid, and costeffective genetic sex-determination. It outperforms existing sexing methods both in terms of sensitivity and minimum required template molecule lengths. Therefore, we feel confident that TriXY will prove to be a reliable addition to the toolbox currently used for sex-typing in forensic genetics and other fields of research.

Parallel-processing continuous-flow device for optimization-free polymerase chain reaction.

A parallel-processing four-station polymerase chain reaction (PCR) device has been developed, which performs continuous-flow PCR without optimization of the annealing temperature. Since the annealing temperature of each station can be controlled independently, the device covers an annealing temperature range of 50-68°C, which is wide enough to perform PCR for any DNA fragment regardless of its optimum annealing condition. This arrangement lets us continuously obtain an amplified amount of a DNA fragment at least from one of the stations. The device consists of four identical cylindrical stations (diameter 20mm, height 55mm). A polytetrafluoroethylene capillary reactor (length 2m, I.D. 100μm, O.D. 400μm) is wound helically up around each station. The whole assembly is designed to minimize the number of heating blocks (for providing temperatures of denaturation, annealing, and extension) to be seven and to shape a compact cube (height 55mm, base 60mm × 60mm). The reproducibility for continuous-flow PCR is reasonably high (run-to-run and station-to-station relative standard deviation of their amplification is lower than 6% and about 4%, respectively). Performance on the optimization-free DNA amplification has been evaluated with four DNA samples with different annealing conditions and product sizes (323, 608, 828, and 1101bp), which has demonstrated that in all cases, PCR is successful at least on one station. In addition, three DNA fragments with different lengths (323, 1101, and 2836 bp) have been successfully amplified in a segmented-flow mode without the carry-over contamination between segments. This result suggests that this device could serve as the PCR module of a continuous-flow high-throughput on-line total DNA analysis system integrating all necessary modules from cell lysis/DNA extraction to PCR product analysis.

Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications

To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10−5U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design.

DNA 교차오염 방지기능이 있는 single-tube nested reverse transcription-polymerase chain reaction을 이용한 돼지생식기호흡기증후군바이러스 유전형 감별진단

In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 <TEX>$TCID_{50}$</TEX>/0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.

Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus.

Tembusu virus (TMUV) is a mosquito-borne flavivirus which threatens both poultry production and public health. In this study we developed a complete open reading frame alignment-based rRT-LAMP method for the universal detection of TUMV. To prevent false-positive results, the reaction was supplemented with uracil DNA glycosylase (UDG) to eliminate carryover contamination. The detection limit of the newly developed UDG-rRT-LAMP for TMUV was as low as 100 copies/reaction of viral RNA and 1 × 100.89 − 1 × 101.55 tissue culture infectious dose/100 μL of viruses. There were no cross-reactions with other viruses, and the reproducibility of the assay was confirmed by intra- and inter-assay tests with variability ranging from 0.22–3.33%. The new UDG-rRT-LAMP method for TMUV produced the same results as viral isolation combined with RT-PCR as the “gold standard” in 96.88% of cases for 81 clinical samples from subjects with suspected TMUV infection. The addition of UDG can eliminate as much as 1 × 10−16 g/reaction of contaminants, which can significantly reduce the likelihood of false-positive results during the rRT-LAMP reaction. Our result indicated that our UDG-rRT-LAMP is a rapid, sensitive, specific, and reliable method that can effectively prevent carryover contamination in the detection of TMUV.