Non-specific Amplification Research Articles

Development of a Diagnostic Assay for Race Differentiation of Podosphaera macularis.

Hop powdery mildew (caused by Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. After 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in the cultivar Cascade. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race(s) may be present in the region and able to infect susceptible hop genotypes. Current methods for determining the races of P. macularis are labor intensive, costly, and typically require more than 14 days to obtain results. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, referred to as V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6 isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short insertion-deletion polymorphisms. Two differentially labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting duplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with different species of powdery mildew fungi and other microorganisms associated with hop. Weak nonspecific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, nonspecification amplification is not a concern when differentiating pathogen race from colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.

Novel Application of Nanofluidic Chip Digital PCR for Detection of African Swine Fever Virus.

African swine fever virus (ASFV) gives rise to a grievous transboundary and infectious disease, African swine fever (ASF), which has caused a great economic loss in the swine industry. To prevent and control ASF, once suspicious symptoms have presented, the movement of animal and pork products should be stopped, and then, laboratory testing should be adopted to diagnose ASF. A method for ASFV DNA quantification is presented in this research, which utilizes the next-generation PCR platform, nanofluidic chip digital PCR (cdPCR). The cdPCR detection showed good linearity and repeatability. The limit of detection for cdPCR is 30.1995 copies per reaction, whereas no non-specific amplification curve was found with other swine viruses. In the detection of 69 clinical samples, the cdPCR showed significant consistency [91.30% (63/69)] to the Office International des Epizooties-approved quantitative PCR. Compared with the commercial quantitative PCR kit, the sensitivity of the cdPCR assay was 86.27% (44/50), and the specificity was 94.44% (17/18). The positive coincidence rate of the cdPCR assay was 88% (44/50). The total coincidence rate of the cdPCR and kit was 89.86% (62/69), and the kappa value reached 0.800 (P < 0.0001). This is the first time that cdPCR has been applied to detecting ASFV successfully.

Scientific instrument for creation of effective Cooper pair mass spectroscopy

We describe electronic instruments for creation of effective Cooper pair spectroscopy. The suggested spectroscopy requires study of electric field effects on the surface of cleaved superconductors. The electronic instrument reacquires low noise amplifier with 106 amplitude amplification which we have formerly used for study of Johnson-Nyquist and Schottky noises. The nonspecific amplifier is followed by high-Q tunable resonance filter based on schematics of general impedance converter topology which is also and innovative device. The work of the device is based on the Manhattan equation of operational amplifier. After a final nonspecific amplification the total amplification can exceed 109 and in such a way sub-nano-volt signals can be reliably detected. In short the observation of new effects in condensed matter physics leads to creation of new generation of electronic equipment.

Development and Clinical Validation of Iso-IMRS: A Novel Diagnostic Assay for P. falciparum Malaria.

In many countries targeting malaria elimination, persistent malaria infections can have parasite loads significantly below the lower limit of detection (LLOD) of standard diagnostic techniques, making them difficult to identify and treat. The most sensitive diagnostic methods involve amplification and detection of Plasmodium DNA by polymerase chain reaction (PCR), which requires expensive thermal cycling equipment and is difficult to deploy in resource-limited settings. Isothermal DNA amplification assays have been developed, but they require complex primer design, resulting in high nonspecific amplification, and show a decrease in sensitivity than PCR methods. Here, we have used a computational approach to design a novel isothermal amplification assay with a simple primer design to amplify P. falciparum DNA with analytical sensitivity comparable to PCR. We have identified short DNA sequences repeated throughout the parasite genome to be used as primers for DNA amplification and demonstrated that these primers can be used, without modification, to isothermally amplify P. falciparum parasite DNA via strand displacement amplification. Our novel assay shows a LLOD of ∼1 parasite/μL within a 30 min amplification time. The assay was demonstrated with clinical samples using patient blood and saliva. We further characterized the assay using direct amplicon next-generation sequencing and modified the assay to work with a visual readout. The technique developed here achieves similar analytical sensitivity to current gold standard PCR assays requiring a fraction of time and resources for PCR. This highly sensitive isothermal assay can be more easily adapted to field settings, making it a potentially useful tool for malaria elimination.

The Development and Optimization of Primer Sets Used to Study the Relative Expression of Androgen Receptor Gene in Turkey (Meleagris gallopavo)

The Androgen Receptor (AR) Gene’s expression is essential during puberty and testes maturation, which is also used as a reference for Turkey’s breeding program. Therefore, this study aims to develop and optimize two primer sets for studying relative expression using qPCR technology. The primers were designed to amplify specific regions in the AR gene as the main target, and the β-actin gene as an internal control. They were tested using in-silico and amplicon sequencing, as well as efficiency calculation with a constructed standard curve from serially diluted reactions. Based on the sequencing methods, the primers amplified the corresponding regions of the respective targets. The primer for AR gene had an efficiency of 98.03%, a slope of -3.37, and an R2 of 0.995, while that of the β-actin gene had an efficiency of 98.01%, a slope of -3.371, and an R2 of 0.999. The two efficiencies exceeded the standard (93 -103%) value and the melting curve analysis showed that no non-specific amplification was discovered for both primers. According to the tests, the primers are suggested as acceptable to be used for the relative expression study of the AR gene in Turkey.

Antigen tests for COVID-19.

PCR diagnosis has been considered as the gold standard for coronavirus disease 2019 (COVID-19) and other many diseases. However, there are many problems in using PCR, such as non-specific (i.e., false-positive) and false-negative amplifications, the limits of a target sample volume, deactivation of the enzymes used, complicated techniques, difficulty in designing probe sequences, and the expense. We, thus, need an alternative to PCR, for example an ultrasensitive antigen test. In the present review, we summarize the following three topics. (1) The problems of PCR are outlined. (2) The antigen tests are surveyed in the literature that was published in 2020, and their pros and cons are discussed for commercially available antigen tests. (3) Our own antigen test on the basis of an ultrasensitive enzyme-linked immunosorbent assay (ELISA) is introduced. Finally, we discuss the possibility that our antigen test by an ultrasensitive ELISA technique will become the gold standard for diagnosis of COVID-19 and other diseases.

Comparison of Seven Commercial TaqMan Master Mixes and Two Real-Time PCR Platforms Regarding the Rapid Detection of Porcine DNA

A pig-specific real-time PCR assay based on the mitochondrial ND5 gene was developed to detect porcine material in food and other products. To optimize the performance of assay, seven commercial TaqMan master mixes and two real-time PCR platforms (Applied Biosystems StepOnePlus and Bio-rad CFX Connect) were used to evaluate the limit of detection (LOD) as well as the PCR efficiency and specificity. The LODs and PCR efficiencies for the seven master mixes on two platforms were 0.5–5 pg/reaction and 84.96%–108.80%, respectively. Additionally, non-specific amplifications of DNA from other animal samples (human, dog, cow, and chicken) were observed for four master mixes. These results imply that the sensitivity and specificity of a real-time PCR assay may vary depending on master mix and platform used. The best combination of master mix and real-time PCR platform can accurately detect 0.5 pg porcine DNA, with a PCR efficiency of 100.49%.

Click-based amplification: designed to facilitate various target labelling modes with ultralow background amplification.

We here describe a fluorescent signal amplification method termed “Click-based amplification” that can be well integrated with various click-labelling modes, including chemical labelling, genetic incorporation and covalent inhibitor probe mediated target labelling. Picolyl azide (pAz) was used as a functional group of a streptavidin-based amplifier to enhance the efficiency of click chemistry. Click-based amplification provided 3.0–12.7 fold amplification on fixed HeLa cells with different click-labelling modes. Click-based amplification has proven to be superior to tyramide signal amplification (TSA) in view of its low nonspecific amplification and high signal-to-noise ratio. Moreover, in terms of the challenging signal amplification of tissue specimens, Click-based amplification successfully achieved remarkable fluorescence enhancement on intestinal tissue slices of afatinib-N3 treated mice, which provided direct evidence of the presence of afatinib-N3 in the intestinal tissues and helped in revealing the off-target toxicity of afatinib. Collectively, these results illustrate that Click-based amplification could serve as a promising method for bioimaging studies.

Optimization of environmental DNA extraction and amplification methods for metabarcoding of deep-sea fish

Analyses of environmental DNA (eDNA) from macroorganisms in aquatic environments have greatly advanced in recent years. In particular, eDNA metabarcoding of fish using universal PCR primers has been reported in various waters. Although pumped deep-sea water was used for eDNA metabarcoding of deep-sea fish, conventional methods only resulted in small amounts of extracted eDNA and subsequent few or no PCR amplicons. To optimize eDNA metabarcoding of deep-sea fish from pumped deep-sea water, we modified conventional procedures of eDNA extraction and PCR amplification. Here, we propose a modified eDNA extraction method, in which a filter used for eDNA sampling was shredded and incubated in microtubes for efficient lysis of eDNA sources. Total eDNA yield extracted using the modified protocol was approximately six-fold higher than that extracted by the conventional protocol. The PCR enzyme Platinum SuperFi II DNA Polymerase successfully amplified a target region of fish universal primers (MiFish) from trace amounts of eDNA extracted from pumped deep-sea water and suppressed nonspecific amplifications more effectively than the enzyme used in conventional methods. Approximately 93% of the sequence reads acquired by next generation sequencing of these amplicons were derived from fish. The improved procedure presented here provided effective eDNA metabarcoding of deep-sea fish.•A modified eDNA extraction protocol, in which a filter was shredded and incubated in microtubes, increased eDNA yields extracted from pumped deep-sea water over the conventional method.•The PCR enzyme Platinum SuperFi II DNA polymerase improved the amplification efficiency of trace amounts of MiFish objectives in eDNA extracted from pumped deep-sea water with suppressing nonspecific amplifications.•The use of Platinum SuperFi II DNA polymerase for eDNA metabarcoding using MiFish primers resulted in the acquisition of abundant sequence reads of deep-sea fish through next generation sequencing.

409. Rapid Development of a Multiplexed PCR Prototype Method that Offers a Syndromic Diagnostic Option by Integrating Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Detection with Twenty-One Other Common Respiratory Pathogens

BackgroundThe US Food and Drug Administration (FDA) has granted Emergency Use Authorization (EUA) for multiple PCR-based tests to aid in the diagnosis and containment of COVID-19. A vast majority of these tests detect only SARS-CoV-2 which causes symptoms similar to those caused by other respiratory pathogens. Hence, other etiologies or co-infections requiring a different therapy may be missed. The prototype BioFire® Respiratory Panel 2.1 (RP2.1) continues the syndromic approach of the FDA-cleared BioFire® Respiratory Panel 2 (RP2), to provide the ability to simultaneously detect 22 common respiratory pathogens, including SARS-CoV-2, from nasopharyngeal swab (NPS) specimens. The goal of this study was to rapidly develop a RP2.1 prototype that contains high-performing SARS-CoV-2 assays and maintains the performance of assays retained from RP2.MethodsTwelve assays designed for four SARS-CoV-2 genes were tested for compatibility with the RP2 assays and conditions. All retained RP2 assays were evaluated to verify established RP2 performance. The sensitivity of novel SARS-CoV-2 assays was estimated with nucleic acids at BioFire and contrived live virus NPS samples at MRIGlobal. Primer homology of SARS-CoV-2 assays to > 15,000 SARS-CoV-2 genomes from accessible databases was assessed for in silico inclusivityResultsA prototype multiplexed PCR panel containing assays for 22 pathogens was developed in a 5-week period. Of the 12 SARS-CoV-2 assays, 7 were compatible with the RP2 conditions; 2 were selected for the prototype. No false positive results due to cross-reactivity with unintended analytes or non-specific amplification in negative samples were observed for any assays. All retained RP2 assays were detected at or near their established LoD. The SARS-CoV-2 LoD was estimated at 103 -102 genomes/mL with both nucleic acid and live virus spiked into NPS. Together, the assays are 100% inclusive for all 15,370 complete SARS-CoV-2 genomes assessed in silico for reactivity.ConclusionThe results of this study indicate a strong potential for RP2.1 to serve as a sensitive comprehensive syndromic option to aid in the diagnosis of COVID-19 as well as respiratory syndromes caused by other pathogens, including co-infections.This study was performed with a test not cleared for diagnostic use.Disclosures All Authors: No reported disclosures

Method for Solving Non-specific Amplification Interference of Fluorescence Quantitative PCR in Gene Detection

Objective: To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection. Method: A three-step method was used for amplification, and the quantitative fluorescence signal collection process was set in the extension stage. Results: Three-step amplification has the advantages of wide application range; improved accuracy; and reduced primer design requirements. Conclusion: The interference of non-specific amplification signals was effectively avoided, the melting curve plotting process was omitted, the reaction time was shortened, and the detection accuracy was improved.

Efficient, Rapid, and Sensitive Detection of Plant RNA Viruses With One-Pot RT-RPA-CRISPR/Cas12a Assay.

Most viruses that infect plants use RNA to carry their genomic information; timely and robust detection methods are crucial for efficient control of these diverse pathogens. The RNA viruses, potexvirus (Potexvirus, family Alphaflexiviridae), potyvirus (Potyvirus, family Potyviridae), and tobamovirus (Tobamovirus, family Virgaviridae) are among the most economically damaging pathogenic plant viruses, as they are highly infectious and distributed worldwide. Their infection of crop plants, alone or together with other viruses, causes severe yield losses. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and others have been harnessed for the detection of DNA- and RNA-based viruses. However, they have a high rate of non-specific amplification and other drawbacks. The collateral activities of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems such as Cas12 and Cas14 (which act on ssDNA) and Cas13 (which acts on ssRNA) have recently been exploited to develop highly sensitive, specific, and rapid detection platforms. Here, we report the development of a simple, rapid, and efficient RT- RPA method, coupled with a CRISPR/Cas12a-based one-step detection assay, to detect plant RNA viruses. This diagnostic method can be performed at a single temperature in less than 30 min and integrated with an inexpensive commercially available fluorescence visualizer to facilitate rapid, in-field diagnosis of plant RNA viruses. Our developed assay provides an efficient and robust detection platform to accelerate plant pathogen detection and fast-track containment strategies.

5-Hydroxymethylcytosine Glucosylation-Triggered Helicase-Dependent Amplification-Based Fluorescent Biosensor for Sensitive Detection of β-Glucosyltransferase with Zero Background Signal

β-glucosyltransferase (β-GT) catalyzes the glucosylation of 5-hydroxymethylcytosine (5-hmC) to enable the survival of bacteriophage and parasite in host cells, and it is a critical tool enzyme for 5-hmC assay. However, few methods are available for β-glucosyltransferase assay, and they usually have the drawbacks of radioactive contamination, high background, laborious procedures, and unsatisfactory sensitivity. Herein, we develop a new fluorescent biosensor with zero background signal for sensitive detection of β-GT activity based on 5-hmC glucosylation-triggered helicase-dependent amplification (HDA). The detection probe we designed may act as both a probe for β-GT recognition and a template for HDA amplification. The β-GT-catalyzed 5-hmC glucosylation can protect the detection probes from both the cleavage by MfeI restrictive enzyme and the digestion by exonucleases I and III. The remaining detection probes can subsequently act as the templates for exponential HDA amplification to generate numerous double-stranded DNA products, which can be easily detected by SYBR Green I in a label-free manner. The zero background can be achieved by efficient elimination of primer-dimer nonspecific amplification and complete digestion of nonglucosylated detection probes. This biosensor exhibits high sensitivity and good specificity, and it can be further used to analyze β-GT kinetic parameters and screen the inhibitors, providing a powerful platform for deeper understanding of β-GT biological functions and promoting β-GT-related epigenetic studies. Furthermore, this biosensor can be extended to detect various DNA-modifying enzymes by simply replacing the recognition sequence and restriction enzyme.

Rapid identification of drug-type and fiber-type cannabis by allele specific duplex PCR

Cannabis is classified into two types: drug-type cannabis, which is abused worldwide, and fiber-type cannabis, which is used for industrial purposes. The two types are a result of differences in the sequences of tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes. In the present study, we aimed to establish a PCR-based method to distinguish between drug-type and fiber-type cannabis by detecting the differences in the sequences of THCAS and CBDAS. We constructed a single-plex PCR targeting active THCAS, and observed drug-type cannabis-specific amplification when using 10pg to 1ng of DNA; however, amplification was also observed in fiber-type cannabis when the DNA content reached 10ng. Similarly, single-plex PCR targeting active CBDAS showed fiber-type cannabis-specific amplification in 100pg of DNA, as well as in >1ng of drug-type cannabis DNA. Therefore, when an allele-specific duplex PCR system was constructed, in which both primer sets were mixed at an appropriate ratio, unintended nonspecific amplification was suppressed and amplicons of different sizes were observed between the drug-type and fiber-type cannabis, using DNA samples in the range of 1pg to 10ng. When the constructed duplex PCR was performed on DNA extracted from various cannabis seed samples, it was possible to distinguish between the drug-type and the fiber-type as well as detect a hybrid-type with both active THCAS and active CBDAS and a special type with neither. The identification method developed in the present study can quickly and accurately distinguish between drug-type and fiber-type cannabis, and is expected to be used for various purposes such as the detection of genetic contamination of industrial hemp as well as forensic examination of cannabis-related cases.

Interleukin-8 Receptors CXCR1 and CXCR2 Are Not Expressed by Endothelial Colony-forming Cells.

Endothelial colony-forming cells (ECFCs) are human vasculogenic cells described as potential cell therapy product and good candidates for being a vascular liquid biopsy. Since interleukin-8 (IL-8) is a main actor in senescence, its ability to interact with ECFCs has been explored. However, expression of CXCR1 and CXCR2, the two cellular receptors for IL-8, by ECFCs remain controversial as several teams published contradictory reports. Using complementary technical approaches, we have investigated the presence of these receptors on ECFCs isolated from cord blood. First, CXCR1 and CXCR2 were not detected on several clones of cord blood- endothelial colony-forming cell using different antibodies available, in contrast to well-known positive cells. We then compared the RT-PCR primers used in different papers to search for the presence of CXCR1 and CXCR2 mRNA and found that several primer pairs used could lead to non-specific DNA amplification. Last, we confirmed those results by RNA sequencing. CXCR1 and CXCR2 were not detected in ECFCs in contrary to human-induced pluripotent stem cell-derived endothelial cells (h-iECs). In conclusion, using three different approaches, we confirmed that CXCR1 and CXCR2 were not expressed at mRNA or protein level by ECFCs. Thus, IL-8 secretion by ECFCs, its effects in angiogenesis and their involvement in senescent process need to be reanalyzed according to this absence of CXCR-1 and - 2 in ECFCs.Graphical Abstract.

End-point RT-PCR: A potential alternative for diagnosing coronavirus disease 2019 (COVID-19)

Real-time reverse transcription-polymerase chain reaction (RT-qPCR) is considered the "gold standard" for the direct diagnosis of SARS-CoV-2 infections. However, routine diagnosis by RT-qPCR is a limitation for many laboratories, mainly due to the infrastructure and/or disproportionate relationship between demand and supply of inputs. In this context, and to increase the diagnostic coverage of SARS-CoV-2 infections, we describe an alternative, sensitive and specific one-step end-point RT-PCR for the detection of the SARS-CoV-2 E gene. The performance of the RT-PCR was evaluated in 43 clinical samples, of which 10 and 33 were previously identified as negative and positive, respectively, by RT-qPCR. Among the positive samples, 15 and 18 were from asymptomatic and symptomatic individuals, respectively. Here, 32/33 of the positive samples in the RT-qPCR, including from asymptomatic individuals, were found positive in the RT-PCR (Ct 15.94–34.92). The analytical sensitivity of the assay was about 7.15–9 copies of vRNA/μL, and nonspecific amplifications were not observed in SARS-CoV-2 negative samples. Importantly, the RT-PCR reactions were performed in a 10 μL final volume. Finally, considering specificity, analytical sensitivity and cost reduction, we believe that the RT-PCR platform described here may be a viable option for the diagnostic of SARS-CoV-2 infections in laboratories in which RT-qPCR is not available.

Authentication of camel meat using species-specific PCR and PCR-RFLP.

In India and some of the African countries, one of the unconventional meats receiving the latest attention in meat adulteration is camel meat. So, the objective of this study was to develop a species-specific PCR based on mitochondrial cytochrome b (CYTB) gene and a PCR-RFLP assay of mitochondrial 12S rRNA to identify camel meat in suspected samples. Known sample of camel meat, samples suspected to be from illegally slaughtered camel and known samples of cattle, buffalo, sheep, goat, pork and chicken were used in the study. DNA were extracted from all samples following spin column method and PCR amplification were carried out using both CYTB and 12S rRNA gene primers. The CYTB gene amplification produced amplicon with a size of 435bp without any non-specific spurious amplification towards other species studied. Further, the 12S rRNA PCR products were analysed both by sequencing and by RFLP using enzyme AluI. On BLAST analysis the 448bp sequence obtained from suspected samples showed > 99% sequence homology to previously reported Camelus dromedaries (accession no: AM 9369251.1). On AluI digestion of the 448bp product from both known and suspected camel samples, a specific RFLP pattern with three distinct products of 90, 148 and 210bp size were evident, which were significantly different from the pattern of cattle, sheep, goat, chicken and buffalo. Further, after in-house validation, this cost effective and rapid method of camel meat identification is placed into practice for regular screening of vetero-legal samples in the lab.

Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis

The targeted sequencing of the 16S rRNA gene is one of the most frequently employed techniques in the field of microbial ecology, with the bacterial communities of a wide variety of niches in the human body have been characterised in this way. This is performed by targeting one or more hypervariable (V) regions within the 16S rRNA gene in order to produce an amplicon suitable in size for next generation sequencing. To date, all technical research has focused on the ability of different V regions to accurately resolve the composition of bacterial communities. We present here an underreported artefact associated with 16S rRNA gene sequencing, namely the off-target amplification of human DNA. By analysing 16S rRNA gene sequencing data from a selection of human sites we highlighted samples susceptible to this off-target amplification when using the popular primer pair targeting the V3–V4 region of the gene. The most severely affected sample type identified (breast tumour samples) were then re-analysed using the V1–V2 primer set, showing considerable reduction in off target amplification. Our data indicate that human biopsy samples should preferably be amplified using primers targeting the V1–V2 region. It is shown here that these primers result in on average 80% less human genome aligning reads, allowing for more statistically significant analysis of the bacterial communities residing in these samples.

Development and application of multiplex PCR assay for the simultaneous detection of Babesia vogeli, Ehrlichia canis and Hepatozoon canis in dogs

A multiplex PCR assay was standardized and evaluated to simultaneously detect the DNA of Babesia vogeli, Ehrlichia canis and Hepatozoon canis in dogs of selected districts of Punjab state, India. Amplicons of 602 bp, 380 bp and 306 bp corresponding to B. vogeli (18S rRNA gene), E. canis (VirB9 gene), and H. canis (18S rRNA gene) were obtained, without any non-specific amplification. The results of multiplex PCR assay were further compared with the corresponding singleplex PCR assay. The diagnostic sensitivity and specificity of multiplex PCR assay with respect to singleplex PCR assay in the detection of B. vogeli, E. canis and H. canis varied from 50% to 100% and 92.08% to 98.79%, respectively revealing “moderate” to “very good” agreement by kappa value statistics. Blood samples from 322 dogs collected from selected districts of Punjab state, India, when screened by microscopy revealed the prevalence of B. vogeli, E. canis and H. canis as 0.31%, 0.93% and 1.86%, respectively whereas with multiplex PCR assay the values were 0.93%, 10.24% and 4.65%, respectively, with concurrent infection of E. canis & H. canis (1.86%) and B. vogeli & E. canis (0.31%). The diagnostic sensitivity and specificity of multiplex PCR assay with respect to microscopy in the detection of B. vogeli, E. canis and H. canis varied from 69.15% to 100% and 85.11% to 92.33%, respectively revealing “fair” agreement by kappa value statistics and the data was statistically significant. The analytical sensitivity of multiplex PCR assay in the detection of B. vogeli, E. canis and H. canis was 100 pg, 10 pg and 0.1 pg, respectively, whereas the values for the singleplex counterpart were 0.1 pg, 0.01 pg and 0.01 pg. Furthermore, various risk factors viz. age, breed, sex, season and districts were non-significantly associated with the prevalence of these haemoparasites except for E. canis that revealed a significant association with districts by multiplex PCR assay. Therefore the multiplex PCR assay developed may be useful in identification of the aetiological agents of these diseases during their early phase, which may in turn be useful in development of better health care and appropriate treatment of suspected dogs, particularly in endemic regions.

Effects of graphene oxide on PCR amplification for microbial community survey

BackgroundGraphene oxide (GO) has been suggested as an efficient assistant additive to eliminate non-specific amplification of the polymerase chain reaction (PCR). Although many studies have focused on exploring its molecular mechanism, the practice of GO on the quantitation of microbial community has not been implemented yet. In this study, GO was added in PCR system to explore the changes on removing typical amplification errors, such as chimera and mismatches on two kinds of mock communities (an evenly mixed and a staggered mock communities) and environmental samples.ResultsHigh-throughput sequencing of bacterial and fungal communities, based on 16S rRNA genes and internal transcribed spacers (ITS) respectively, showed that GO could significantly increase large segmental error (chimeric sequence) in PCR procedure while had no specific effect on point error (mismatched sequence). Besides, GO reduced the α-diversity of community, and changed the composition of fungal community more obviously than bacterial community.ConclusionsOur study provides the first quantitative data on microbial community level to prove the negative effect of GO, and also indicates that there may be a more complex interaction between GO and comprehensive DNA fragments in PCR process.