Detection of Clostridium botulinum neurotoxin type A using immuno-PCR

AIMS: An immuno-polymerase chain reaction (immuno-PCR) has been developed for the sensitive detection of antigens, which greatly extends the detection limits of immunoassays. In the current study, the method was applied to the detection of Clostridium botulinum neurotoxin type A (BTx-A). METHODS AND RESULTS: Anti-BTx-A antibody-DNA conjugates were synthesized using a heterobifunctional cross-linker reagent to covalently link the reporter DNA and the antibodies. The antibody-DNA conjugates with antigens were amplified by PCR, and dose-dependent relationships for each analyte were demonstrated. Detection limits of immuno-PCR for BTx-A (3.33 x 10(-17) mol) exceeded the conventional enzyme-linked immunosorbent assay (3.33 x 10(-14) mol) by a 1000-fold enhancement in detection sensitivity. CONCLUSION: Detection of BTx-A antigens by immuno-PCR demonstrated 100% sensitivity and 100% specificity in 100-fold magnitude below the detection limit of ELISA. SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that the immuno-PCR method could be used to detect a very low level of BTx-A for clinical diagnosis.     

IQPA: Isothermal nucleic acid amplification-based immunoassay using DNAzyme as the reporter system

Immunoassays are often coupled to peroxidase activity for antigen detection. Sensitivity and speed of detection has been increased by the advent of hybrid methods such as immuno-PCR (polymerase chain reaction). However, a more simplified immunoassay that retains both colorimetric peroxidase detection and effective DNA amplification in a setting closer to field application conditions has been nonexistent. Here we describe a method that successfully combines a competitive immunoassay with the new isothermal quadruplex-primed amplification (QPA) to generate excess quadruplex reporter molecules with intrinsic peroxidase DNAzyme activity.     

A highly sensitive immuno-PCR assay for detection of H5N1 avian influenza virus

With an aim at detecting the ultra-low concentration of avian influenza virus (AIV), a highly sensitive hybrid assay based on immunology and polymerase chain reaction was developed. The TopYield microtiter plates were coated with ten-fold serial dilutions of H5N1 subtype AIV ranging from 10 EID₅₀ ml⁻¹~10⁻⁴ EID₅₀ ml⁻¹,which was recognized by mouse anti-AIV H5 monoclonal antibody (MAb) that was directly linked with reporter DNA using a heterobifunctional cross-linker. After extensive washing, the reporter DNA including a BamH I-restriction site was released by a specific enzymatic restriction, then transferred to PCR tubes, amplified, and used as the signal for detection of AIV. Under the optimized condition, MAb-based immuno-PCR (IPCR) method could measure 100 μl of AIV H5N1 with 10⁻⁴ EID₅₀ ml⁻¹.To evaluate the sensitivity of IPCR, the same concentration and volume of AIV H5N1 were detected by conventional RT–PCR and sandwich ELISA. The results showed that IPCR had an approximately 1,000-fold improvement over the conventional ELISA, and a 100-fold enhancement compared with RT–PCR in detection sensitivity. To further evaluate the specificity of IPCR for AIV H5 subtype, the tracheal swab specimens, taken from chickens which were infected with H9N2, and the allantoic fluid from eggs inoculated by AIV H3N2, H7N1, H9N2, were detected by IPCR. To mimic clinical samples, pharyngeal–tracheal swab specimens were collected from healthy chickens and spiked with H5N1, H5N2, H5N3 for analysis by immuno-PCR. The results demonstrated that IPCR was a highly sensitive and specific assay for AIV H5, and could be applied to clinical detection for low amount of AIV H5 subtype. This MAb-based immuno-PCR method provided a platform capable of mass screening of clinical samples for AIV H5 subtype and could serve as a model for other immuno-PCR assays.     

Real-time immuno-polymerase chain reaction in a 384-well format: Detection of vascular endothelial growth factor and epidermal growth factor-like domain 7

Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody–DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51 pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.     

A novel strategy for construction of immuno-PCR gene probe and its preliminary application in diagnosis

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A dual-luciferase reporter system for studying recoding signals.

A new reporter system has been developed for measuring translation coupling efficiency of recoding mechanisms such as frameshifting or readthrough. A recoding test sequence is cloned in between the renilla and firefly luciferase reporter genes and the two luciferase activities are subsequently measured in the same tube. The normalized ratio of the two activities is proportional to the efficiency with which the ribosome "reads" the recoding signal making the transition from one open reading frame to the next. The internal control from measuring both activities provides a convenient and reliable assay of efficiency. This is the first enzymatic dual reporter assay suitable for in vitro translation. Translation signals can be tested in vivo and in vitro from a single construct, which allows an intimate comparison between the two systems. The assay is applicable for high throughput screening procedures. The dual-luciferase reporter system has been applied to in vivo and in vitro recoding of HIV-1 gag-pol, MMTV gag-pro, MuLV gag-pol, and human antizyme.     

General and hybrid correlation nuclear magnetic resonance analysis of phosphorus in Phytophthora palmivora

As a specific tumor marker, prostate-specific antigen (PSA) is widely used for the early diagnosis of prostate cancer. Sensitive and specific methods are required to improve the diagnostic accuracy of PSA detection. In the current study, we compared the immuno-polymerase chain reaction (immuno-PCR) method with the solid-phase proximity ligation assay (SP-PLA) with respect to the detection of PSA. Using oligonucleotide-labeled antibody probes, we used both immuno-PCR and SP-PLA to detect trace levels of PSA. The nucleic acid sequences can be monitored using real-time PCR. SP-PLA, however, was found to be superior in terms of both the detection limit and the dynamic range. To detect even lower levels of PSA, we used the loop-mediated isothermal amplification (LAMP) method to measure the levels of reporter DNA molecules in SP-PLA. The sensitivity of the LAMP method is 0.001 pM, which is approximately 100-fold higher than the sensitivities of the other assays. The results suggest that an SP-PLA- and LAMP-based protocol with oligonucleotide-labeled antibody probes may have great application in detecting PSA or other proteins present at trace levels.     

PEPCK不同启动子调控报告基因表达效率的比较

目的构建两种不同的磷酸烯醇式丙酮酸羧激酶（PEPCK）基因启动子调控报告基因表达质粒,比较该启动子在各种细胞系中的表达效率。方法采用PCR克隆大鼠PEPCK启动子1323 bp和560 bp两条片段,将两个PEPCK启动子片段插入pGL4.26-Luc2报告质粒中,将重组质粒转染到肝脏细胞系和脂肪细胞系,以非脂肪或肝脏细胞系做对照,比较PEPCK驱动荧光素酶表达效率。结果通过酶切鉴定及基因测序,证明pGL4.26-PEPCK-Luc2荧光素酶表达质粒构建成功,且能够在体外表达荧光素酶。结论两种同片段的PEPCK启动子在各细胞系中的表达效率差异无显著性。     

Immuno-PCR--a new tool for paleomicrobiology: the plague paradigm

Background

The cause of past plague pandemics was controversial but several research teams used PCR techniques and dental pulp as the primary material to reveal that they were caused by Yersinia pestis. However, the degradation of DNA limits the ability to detect ancient infections.

Methods

We used for the first time immuno-PCR to detect Yersinia pestis antigens; it can detect protein concentrations 70 times lower than the standard ELISA. After determining the cut-off value, we tested 34 teeth that were obtained from mass graves of plague, and compared previous PCR results with ELISA and immuno-PCR results.

Results

The immuno-PCR technique was the most sensitive (14 out of 34) followed by the PCR technique (10 out of 34) and ELISA (3 out of 34). The combination of these three methods identified 18 out of 34 (53%) teeth as presumably being from people with the plague.

Conclusion

Immuno-PCR is specific (no false-positive samples were found) and more sensitive than the currently used method to detect antigens of ancient infections in dental pulp. The combination of three methods, ELISA, PCR and immuno-PCR, increased the capacity to identify ancient pathogens in dental pulp.     

Detection of bacterial antigens using immuno-PCR

E. KAKIZAKI, T. YOSHIDA, H. KAWAKAMI, M. OSETO, T. SAKAI AND M. SAKAI. 1996. A new and very sensitive antigen detection technique, immuno-polymerase chain reaction (immuno-PCR), was developed. This method is basically similar to the enzyme-linked immunosorbent assay which detects an antigen-antibody reaction, but instead of an enzyme being conjugated to an antibody, a DNA fragment is used and this DNA can be amplified by PCR. We applied this method to the detection of the fish pathogen, Pasteurella piscicida , in naturally infected yellowtail. Using immuno-PCR, 3.4 cfu ml⁻¹ of bacteria could be detected. In comparison, ELISA detected only 3.4 × 10⁴ cfu ml⁻¹. Immuno-PCR is a powerful method for detection of pathogens in host tissues.     

EphA3基因启动子区域定位表达及活性分析

目的构建含有不同长度EphA3基因启动子片段的报告基因载体,研究其在293T细胞和MEF细胞中的转录活性。方法以Balb／C小鼠基因组DNA为模板,扩增不同长度的EphA3基因启动子片段,并克隆进入荧光素酶报告基因质粒pGL3-Basic真核表达载体内。酶切鉴定及基因测序无误后,将重组质粒和pRL—CMV内对照质粒共转染293T和MEF细胞,分析不同长度的OhA3基因启动子片段的转录活性。结果酶切和测序鉴定表明表达载体构建成功,EphA3基因的核心启动子区域位于-279bp～＋110bp之间,在293T细胞和MEF细胞中其转录活性相似。结论成功构建了荧光素报告基因重组质粒,并确定了BphA3基因的核心启动子区域。     

表达猴免疫缺陷病毒Env蛋白的重组鸡痘病毒的构建和筛选

目的:构建并筛选表达猴免疫缺陷病毒(SIV)Env蛋白的重组鸡痘病毒(FPV),并对其进行鉴定。方法:设计引物,通过PCR技术扩增SIV env基因,将其连接到pMD18-T载体上,测序正确后将其克隆入本实验室自行构建的FPV穿梭载体pTKET中,获得重组质粒pTKET-SIV env,然后将其与FPV282E4株共转染原代鸡胚成纤维细胞进行同源重组,以增强型绿色荧光蛋白为筛选标记,通过噬斑筛选获得重组病毒,应用PCR、RT-PCR、Western印迹对重组病毒进行鉴定和遗传稳定性分析。结果:通过10次噬斑筛选,PCR检测表明目的基因已整合到重组FPV基因组中,RT-PCR、Western印迹结果表明SIV Env蛋白在感染细胞内表达且具有抗原性;连续传代20次,PCR、RT-PCR、Western印迹均能检测到外源基因的整合、转录和表达,且未能扩增出FPV-TK基因,表明重组病毒遗传稳定性良好,且病毒已经纯化。结论:获得表达SIV Env蛋白的重组FPV,为进一步免疫试验研究奠定了基础。     

表达HIV-1 gag重组鸡痘病毒的构建与筛选

构建并筛选表达HIV-1 gag蛋白的重组鸡痘病毒,并对其进行鉴定。首先设计引物通过PCR技术扩增HIV-1 gag基因,将其连接到pMD18-T载体上,测序正确后将其克隆入本实验室自行构建的鸡痘病毒穿梭载体pTKET中,获得重组质粒pTKET-HIV gag。然后将其与鸡痘病毒FPV282E4株共转染原代鸡胚成纤维细胞(CEF)进行同源重组,以增强型绿色荧光蛋白(EGFP)为筛选标记,通过噬斑筛选获得重组病毒,应用PCR,RT-PCR,Western blot方法对重组病毒进行鉴定和遗传稳定性分析。结果:通过10次噬斑筛选,PCR检测表明目的基因已整合到重组鸡痘病毒基因组中,RT-PCR,Western blot结果表明HIV-1 gag在感染细胞内成功表达且具有抗原性。连续传代20次,PCR,RT-PCR,Western blot均能检测到外源基因的整合、转录和表达,且未能扩增出FPV-TK基因,表明重组病毒遗传稳定性良好,而且病毒已经纯化。结论:成功获得表达HIV-1gag的重组鸡痘病毒,为进一步免疫试验研究奠定基础。     

A transgene containing lacZ is expressed in primary sensory neurons in zebrafish.

In order to screen for developmentally active chromosomal domains during zebrafish embryogenesis, we generated transgenic fish by microinjecting two different lacZ reporter constructs into fertilized eggs. Transgenic fish were screened among the progeny of injected fish (F0) crossed to non-injected fish. Groups of 15 to 20 progeny of each cross were tested for lacZ expression and/or transmission of injected sequences using PCR and Southern hybridizations. Progeny from 2 of 102 fish injected with supercoiled constructs containing Rous sarcoma virus promoter sequences showed apparently spatially regulated beta-galactosidase (beta-Gal) activity. However, we were not able to detect this reporter construct in DNA from fins of F1 fish. Injections of a linear reporter construct containing mouse heat-shock promoter sequences revealed transmission of injected sequences to F1 progeny in about 6% of cases (8 of 129 fish, tested with PCR). We found one lacZ-expressing line that showed a spatially and temporally restricted expression of lacZ and, therefore, features typical characteristics of "enhancer trap" lines. In this line, lacZ expression starts at 16 hours post-fertilization in trigeminal ganglion cells. At about 24 hours lacZ expression can be detected in trigeminal ganglion neurons and Rohon-Beard neurons, indicating that the development of these two cell types shows common features. The reporter gene has integrated as a single copy. The founder fish was mosaic: 19% of its offspring (3 of 16 tested animals) carried the reporter construct in their fins; about 51% (13 of 27 tested animals) of the progeny of F1 fish were beta-Gal positive indicating full hemizygosity.(ABSTRACT TRUNCATED AT 250 WORDS)     

免疫-PCR法检测梅毒螺旋体特异性抗体

以梅毒螺旋体重组蛋白为抗原,应用免疫-PCR方法检测梅毒螺旋体抗体,并同常规ELISA法进行比较,探讨免疫-PCR方法检测梅毒螺旋体特异性抗体的可行性。结果免疫-PCR法敏感性是常规ELISA法的104倍,阳性检出率高于ELISA法;对照血清标本梅毒螺旋体抗体检测为阴性。表明免疫-PCR方法具有较高敏感性和特异性,有一定的临床推广价值,对梅毒患者的早期诊断及时治疗等具有重要意义。     

解偶联蛋白UCP1启动子荧光素酶报告基因载体的构建

目的:构建解偶联蛋白UCP1启动子荧光素酶报告基因载体,为寻找调控UCP1表达的小分子化合物提供有效工具。方法:从小鼠基因组DNA中PCR扩增小鼠UCP1启动子上游2000 bp序列,并将该序列连接到荧光素酶报告基因载体p GL3-basic中,构建p GL3-UCP1启动子。测序正确后,提取质粒,然后将上述载体与p RL-TK载体共转染至HEK293细胞、小鼠白色脂肪前体细胞和小鼠棕色脂肪前体细胞,48 h后裂解细胞检测荧光素酶的活性。结果:通过PCR成功扩增获得了目的片段,并将其克隆至p GL3-basic中。与细胞内源UCP1表达水平相似,荧光素酶报告系统表明构建的p GL3-UCP1在棕色脂肪细胞中启动子活性最高,在白色脂肪细胞中活性较低,在HEK293细胞中基本没有活性。同时β3肾上腺素受体激动剂CL 316,243同样能够上调p GL3-UCP1的启动子活性。结论:成功构建了小鼠UCP1启动子荧光素酶报告基因载体,并证明在棕色脂肪细胞中,该启动子具有很强的启动子活性,而在白色脂肪和HEK293细胞中,启动子活性很低。该启动子报告系统有望为寻找激活UCP1的小分子化合物提供重要平台。     

Mutational Analysis of Pre-mRNA Splicing in Saccharomyces Cerevisiae Using a Sensitive New Reporter Gene, Cup1

We have developed a new reporter gene fusion to monitor mRNA splicing in yeast. An intron-containing fragment from the Saccharomyces cerevisiae ACT1 gene has been fused to CUP1, the yeast metallothionein homolog. CUP1 is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner. By inserting previously characterized intron mutations into the fusion construct, we have established that the efficiency of splicing correlates with the level of copper resistance of these strains. A highly sensitive assay for 5' splice site usage was designed by engineering an ACT1-CUP1 construct with duplicated 5' splice sites; mutations were introduced into the upstream splice site in order to evaluate the roles of these highly conserved nucleotides in intron recognition. Almost all mutations in the intron portion of the 5' consensus sequence abolish recognition of the mutated site, while mutations in the exon portion of the consensus sequence have variable affects on cleavage at the mutated site. Interestingly, mutations at intron position 4 demonstrate that this nucleotide plays a role in 5' splice site recognition other than by base pairing with U1 snRNA. The use of CUP1 as a reporter gene may be generally applicable for monitoring cellular processes in yeast.     

Phage display mediated immuno-PCR

Immuno-PCR (IPCR) is a powerful detection technology in immunological study and clinical diagnosis due to its ultrasensitivity. Here we introduce a new strategy termed phage display mediated immuno-PCR (PD-IPCR). Instead of utilization of monoclonal antibody (mAb) and chemically bond DNA that required in the conventional IPCR, a recombinant phage particle is applied as a ready reagent for IPCR experiment. The surface displayed single chain variable fragment (scFv) and phage DNA themselves can directly serve as detection antibody and PCR template, respectively. The aim of the design is to overcome shortcoming of low detection sensitivity of scFv so as to largely facilitate the real application of scFv in immunoassay. The idea has been demonstrated by applying hantaan virus nucleocapsid protein (NP) and prion protein (PrP) as detection targets in three experimental protocols (indirect, sandwich and real-time PD-IPCR assays). The detection sensitivity was increased 1000- to 10 000-folds compared with conventional enzyme-linked immunosorbent assays (ELISAs). This proof-of-concept study may serve as a new model to develop an easy to operate, low cost and ultrasensitive immunoassay method for broad applications.     

Restriction site-free insertion of PCR products directionally into vectors

A restriction site-free cloning method has been developed for inserting a PCR product into a vector flexibly and precisely at any desired location with high efficiency. The method uses a pair of DNA integration primers with two portions. The 3' portion isolates the inserts by PCR, and the 5' portion integrates the PCR products into the homologous region of the vector. For mutagenesis, a third portion of mutation-generating sequences can be placed in between the 3' and 5' portions. This method has been used to clone the E. coli gene that codes for peptidyl-tRNA hydrolase, expressing it as a native protein and as a glutathione S-transferase fusion protein. It was also applied to convert a construct of the E. coli fatty acid biosynthesis protein with an N-terminal hexa-histidine tag into a construct with a C-terminal hexa-histidine tag.