Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect’s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.     

六种弧菌多重PCR快速检测方法的建立

目的:检测鳗弧菌、哈氏孤菌、副溶血孤菌、溶藻胶弧菌、霍乱弧菌和创伤孤菌六种水产常见病原菌.方法:以toxR-toxS为靶基因设计引物,建立了一种多重PCR( multiplex PCR,mPCR)快速检测方法.结果:本研究设计的mPCR引物特异性强,与其他弧菌及非孤菌无交叉反应,每次反应的敏感性为10-100 CFU(cell forming unit)/每个反应.结论:应用建立的mPCR方法结果稳定可靠,有望成为检测病害弧菌的有效工具.     

六种弧菌多重PCR快速检测方法的建立

目的:检测鳗弧菌、哈氏弧菌、副溶血弧菌、溶藻胶弧菌、霍乱弧菌和创伤弧菌六种水产常见病原菌。方法:以toxR-toxS为靶基因设计引物,建立了一种多重PCR(multiplex PCR,mPCR)快速检测方法。结果:本研究设计的mPCR引物特异性强,与其他弧菌及非弧菌无交叉反应,每次反应的敏感性为10-100 CFU(cell forming unit)/每个反应。结论:应用建立的mPCR方法结果稳定可靠,有望成为检测病害弧菌的有效工具。     

A Multiplex PCR Method for Rapid Identification of Brachionus Rotifers

Cryptic species are increasingly being recognized in many organisms. In Brachionus rotifers, many morphologically similar yet genetically distinct species/biotypes have been described. A number of Brachionus cryptic species have been recognized among hatchery strains. In this study, we present a simple, one-step genetic method to detect the presence of those Brachionus sp. rotifers that have been found in hatcheries. With the proposed technique, each of the B. plicatilis sensu stricto, B. ibericus, Brachionus sp. Nevada, Brachionus sp. Austria, Brachionus sp. Manjavacas, and Brachionus sp. Cayman species and/or biotypes can be identified with polymerase chain reaction (PCR) analysis. Based on 233 cytochrome c oxidase subunit I sequences, we reviewed all the available cryptic Brachionus sp. genetic polymorphisms, and we designed six nested primers. With these primers, a specific amplicon of distinct size is produced for every one of the involved species/biotypes. Two highly sensitive protocols were developed for using the primers. Many of the primers can be combined in the same PCR. The proposed method has been found to be an effective and practical tool to investigate the presence of the above six cryptic species/biotypes in both individual and communal (bulk) rotifer deoxyribonucleic acid extractions from hatcheries. With this technique, hatchery managers could easily determine their rotifer composition at the level of cryptic species and monitor their cultures more efficiently. Kalliopi Vasileiadou and Spiros Papakostas contributed equally to this work.     

Multiplex PCR for Identification of Bacterial Pathogens of Infectious Pneumonia

Russian Journal of Bioorganic Chemistry - A multiplex PCR system has been developed and optimized for rapid detection of the five main pathogens of bacterial pneumonia. The system can be expanded...     

Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.     

多重PCR技术在病原检测中的应用

多重PCR能够同时扩增不同片段,具有普通PCR方法不可比拟的优势。简要论述了多重PCR在细菌病原体的检测、病毒病原体的检测、支原体和衣原体及寄生虫的检测、微生物耐药性检测等方面的应用,分析了多重PCR的影响因素及条件优化,并进一步综述了多重PCR的应用前景。     

多种食源性致病菌检测的多重PCR 方法的研究

目的:利用多重PCR技术,建立可以同时检测多种食源性致病菌的多重PCR方法。方法:分别选择沙门氏菌invA基因,志贺氏菌的ipaH基因,单核细胞增生李斯特氏菌的hlyA基因,大肠杆菌O157:H7的eaeA基因,副溶血弧菌的toxR基因,设计多重PCR引物,建立多重PCR检测体系,并对该体系进行特异性和灵敏度实验。结果:通过对19株菌株进行实验,所有的目标菌株均为阳性,而其余菌株为阴性。对多重PCR体系的灵敏度进行考察,沙门菌的灵敏度为5000 CFU/mL;志贺氏菌的灵敏度为5500CFU/mL;单核细胞增生李斯特氏菌的灵敏度为5200 CFU/mL;O157:H7的灵敏度为5000CFU/mL;副溶血弧菌的灵敏度为6300CFU/mL。结论:建立的多重PCR体系能实现多种致病菌同时检测。     

啮齿动物螺杆菌多重PCR检测方法的建立

目的建立一种可同时检测肝、胆汁、啮齿类三种螺杆菌的多重PCR方法。方法根据已公布的肝、胆汁、啮齿类三种螺杆菌16SrRNA基因序列设计三对特异性引物进行多重PCR并对反应条件进行优化。结果三对引物能分别扩增出特异性的417 bp、364 bp、324 bp目的条带。最佳退火温度为52℃,镁离子浓度为2.0mmol/L,dNTP浓度为200μmol/L,引物浓度为0.625μmol/L。在此条件下多重PCR同时检测的肝、胆汁、啮齿类三种螺杆菌敏感度均为10 fg。结论本实验建立的多重PCR是一种敏感、特异、高效的方法,为同时检测啮齿动物中肝、胆汁、啮齿类三种螺杆菌奠定了良好的基础。     

Multiplex PCR and Next Generation Sequencing for the Non-Invasive Detection of Bladder Cancer

Background

Highly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA.

Methods

DNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free).

Results

Despite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity).

Conclusion

This simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer.     

利用微卫星多重PCR技术鉴定剑尾鱼RR-B系

目的建立多重PCR技术鉴定选育剑尾鱼RR-B系的方法。方法根据可明显鉴定剑尾鱼RR-B系的6对特异性微卫星引物Msa012、Msa014、Msa033、Msb025、Msd003、Msd051,采用不同的组合方式对RR-B系剑尾鱼和红眼红体的非选育剑尾鱼进行多重PCR扩增。结果引物组合1(Msa014、Msb025、Msd003)和组合2(Msa012、Msa033、Msd051)两个三重PCR反应体系能清楚的扩增各个微卫星座位,且各目的片段之间无干扰,易于识别,引物组合1的排除概率为99.98%,引物组合2的排除概率为99.96%,能准确鉴定剑尾鱼RR-B系。结论本检测方法具有较高的稳定性,可快速准确鉴定剑尾鱼RR-B系。     

Detection of Microbial Pathogens in Shellfish with Multiplex PCR

Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl₂ and primer annealing temperature of 55°C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelex™ 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was ≤10¹–10² cells following a “double” multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were subjected to a colorimetric GeneComb™ (BioRad) DNA-DNA hybridization assay. This assay was rapid and showed sensitivity of detection comparable to the agarose gel electrophoresis method. The colorimetric GeneComb™ assay avoids use of hazardous materials inherent in conventional gel electrophoresis and radioactive-based hybridization methods. Multiplex PCR amplification, followed by colorimetric GeneComb™ DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect microbial pathogens in shellfish. Received: 17 November 1997 / Accepted: 17 February 1998     

A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.     

Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas

Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays.Phytoplasmas are wall-less bacteria of the class Mollicutes that inhabit plant phloem and insect vectors. They are known to cause disease in hundreds of plant species worldwide and are classified into 16Sr groups and “Candidatus Phytoplasma” species based on their 16S rRNA gene sequences (14, 21). Since it is not possible to isolate and study phytoplasmas in pure cultures, conventional PCR has become the method of choice for detection and diagnosis. Numerous PCR primer combinations have been devised to amplify the 16S rRNA gene for diagnostics; some of these are universal primers that work on DNA from all phytoplasma phylogenetic groups, while others are group specific (8, 10, 17). However, diagnostics based on these primers can be problematic, with occasional false positives through amplification of other bacteria that might be present in samples (11).Real-time PCR assays have also been developed for both generic and specific phytoplasma detection. In general, the aims have been to produce very specific and sensitive assays for detection of a group-specific nature. For example, TaqMan and SYBR green chemistries have been applied in various diagnostic assays for 16SrX group (1, 3, 18), 16SrV and -XII group (2, 9, 15), 16SrI group (16, 20), and 16SrVI group (5) phytoplasmas. However, several of these specific assays cross-react with phytoplasmas from other groups (5, 16). The most successful attempt to develop a fully universal assay has been a TaqMan assay that was demonstrated to amplify all 16Sr groups except 16SrIV, -XIII, and -XIV, which were not tested (4).     

Detection and Identification of Yersinia pestis by Polymerase Chain Reaction (PCR) Using Multiplex Primers

A PCR method for detection of Yersinia pestis-virulence determinants by the use of multiplex primers was developed. Four pairs of oligonucleotide primers were designed from each gene of three kinds of virulent plasmids and a chromosomal DNA; 60-Md plasmid-located gene (caf1) encoding Y. pestis-specific capsular antigen fraction 1, a Y. pestis-specific region of a yopM gene encoded on 42-Md virulent plasmid, a plasminogen activator gene (pla) encoded on Y. pestis-specific 7-Md plasmid and an invasin protein gene (inv) encoded on chromosomal DNA. This multiplex-primer system was specific for the detection of Y. pestis among pathogenic Yersinia species and other enterobacteriaceae having antigens common to Y. pestis. Since this method is simple and safe, it will be useful to identify and confirm Y. pestis in cases of emergency and for the surveillance of epidemics.     

Modified Multiplex PCR Methods for Comprehensive Detection of Pectinatus and Beer-Spoilage Cocci

Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beer-spoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beer-spoilage species of Pectinatus and cocci, including new species.