Multiplex polymerase chain reaction method for detection of bovine materials in foodstuffs

Rapid identification of bovine materials in animal foodstuffs is essential for effective control of a potential source of bovine spongiform encephalophathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a bovine-specific genomic DNA sequence in foodstuffs. Simultaneously the assay assessed the DNA quality of the experiment system by amplification of a highly conserved eucaryotic DNA region of the 18-S ribosomal gene, helping to check the reliability of the test result. The amplified bovine-specific PCR product was a genomic DNA fragment of lactoferrin, a low copy gene that was different from a commonly used bovine-specific mitochondria sequence for identification of bovine materials. The specificity of this method was confirmed by the absence of detectable homologous PCR product using reference foodstuff samples that lacked bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method could detect the presence of bovine material in foodstuffs when the samples contained > 0.02% bovine-derived meat and bone meal. Furthermore, it was not affected by prolonged heat treatment. The specificity, convenience, and sensitivity of this method suggest that it can be used for the routine detection of bovine-derived materials.     

螺旋通道微流控PCR芯片连续自动扩增DNA片段的研究

研制了由内向外流动的螺旋通道微流控PCR玻璃芯片,减少了PCR反应液在微通道中流动时的分散和阻力;讨论了扩增循环数和进样速度对长片段基因扩增的影响,在26min内成功扩增了质量浓度仅为10ng/mL的6012bpλ-DNA;通过将小孔径石英毛细管作为顺序注射(SI)系统的连接管路,使其死体积降到0.30μL.实现了微升级样品的自动换样、连续PCR扩增和微通道洗涤等功能.样品间无交叉污染.每小时可扩增500bpλ-DNA试样7个.扩增产物片段大小和荧光强度的相对标准偏差分别为0.4%和6.7%.     

Polymerase chain reaction method to detect canis materials by amplification of species-specific DNA fragment

Rapid identification of mammal materials in feeding stuffs and food is essential for effective control of a potential source of pathogens, such as those that cause bovine spongiform encephalopathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a canis-specific mitochondrial DNA sequence in foodstuffs and food. The amplified canis-specific PCR product was a 213 base pair band from the D-loop DNA fragment of mitochondria, a high copy gene which should improve the possibility of amplifying template molecules of adequate size among the degraded DNA fragments brought about by heat denaturation. The specificity of this method was confirmed by 8 canis blood DNA samples (from different breeds of dog) and 9 noncanis animal blood DNA samples (bovine, sheep, porcine, chicken, fish, donkey, rabbit, deer, horse). This method was able to detect the presence of canis material in foodstuffs and in food mixtures even when the concentration of canis-derived meat was reduced to 0.05%. Furthermore, it did not appear to be affected by prolonged heat treatment. This method was developed for detection of canis materials in feeding stuffs, and occasionally for medical jurisprudence detection of canis-derived materials.     

An efficient unnatural base pair for PCR amplification

Expansion of the genetic alphabet by an unnatural base pair system provides a powerful tool for modern biotechnology. As an alternative to previous unnatural base pairs, we have developed a new pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitropyrrole (Pn), which functions in DNA amplification. Pn more selectively pairs with Ds in replication than another previously reported pairing partner, pyrrole-2-carbaldehyde (Pa). The nitro group of Pn efficiently prevented the mispairing with A. High efficiency and selectivity of the Ds-Pn pair in PCR amplification were achieved by using a substrate mixture of the gamma-amidotriphosphate of Ds and the usual triphosphates of Pn and the natural bases, with Vent DNA polymerase as a 3' to 5' exonuclease-proficient polymerase. After 20 cycles of PCR, the total mutation rate of the Ds-Pn site in an amplified DNA fragment was approximately 1%. PCR amplification of DNA fragments containing the unnatural Ds-Pn pair would be useful for expanded genetic systems in DNA-based biotechnology.     

An efficient site-directed mutagenesis using polymerase chain reaction

We report here a simple and efficient method for site-directed mutagenesis using polymerase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restriction gene, we made two point mutations. While one created a new SalI site prior to the SD sequence, the other replaced Glu144 with Lys. A 1.5 kb SalI-PstI fragment isolated from pER101 was used as the template. Two 25 mer oligonucleotide primers containing the desired mutations were synthesized and used to direct PCR amplification with Taq DNA polymerase. About 0.5 microgram of the 0.49 kb fragment was obtained from 0.05 microgram of the 1.5 kb fragment by carrying out polymerase chain reaction for 30 cycles. As calculated theoretically, 99% of the product contained the desired mutations. The product was cloned into pUC19 using SalI and PstI, two of the transformed colonies were randomly chosen for sequence analysis, and both of them were shown to contain the desired mutations. Finally, the amplified fragment was cloned into pER304 to place the EcoRI (Lys144) gene directly under the control of the lambda PL promoter.     

Molecular detection of Burkholderia cepacia in toiletry, cosmetic, and pharmaceutical raw materials and finished products

A polymerase chain reaction (PCR) assay was developed and compared with standard methods for rapid detection of Burkholderia cepacia, a major industrial contaminant, in cosmetic and pharmaceutical raw materials and finished products. Artificially contaminated samples were incubated for 24 h in trypticase soy broth containing 4% Tween 20 and 0.5% soy lecithin. DNA was extracted from each sample using a proteinase K-tris-EDTA-Tween 20 treatment at 35 degrees C. The extracted DNA was added to Ready-To-Go PCR beads and specific DNA primers for B. cepacia. The B. cepacia DNA primers coded for a 209-base pair (bp) fragment of the 16S rRNA ribosomal gene. No DNA amplification was observed in samples that were not spiked with B. cepacia. However, all contaminated samples showed the specific 209-bp fragment for B. cepacia. There was a 100% correlation between standard methods and the PCR assay. Standard microbiological methods required 5-6 days for isolation and identification of spiked microorganisms, whereas PCR detection and identification was completed in 27 h. PCR detection of B. cepacia allows for rapid quality evaluation of cosmetic and pharmaceutical raw materials and finished products.     

Detection of Ovine or Bovine Milk Components in Commercial Camel Milk Powder Using a PCR-Based Method

Food ingredient adulteration, especially the adulteration of milk and dairy products, is one of the important issues of food safety. The large price difference between camel milk powder, ovine, and bovine milk powder may be an incentive for the incorporation of ovine and bovine derived foods in camel milk products. This study evaluated the use of ordinary PCR and real-time PCR for the detection of camel milk powder adulteration based on the presence of ovine and bovine milk components. DNA was extracted from camel, ovine, and bovine milk powder using a deep-processed product column DNA extraction kit. The quality of the extracted DNA was detected by amplifying the target sequence from the mitochondrial Cytb gene, and the extracted DNA was used for the identification of milk powder based on PCR analysis. In addition, PCR-based methods (both ordinary PCR and real-time PCR) were used to detect laboratory adulteration models of milk powder using primers targeting mitochondrial genes. The results show that the ordinary PCR method had better sensitivity and could qualitatively detect ovine and bovine milk components in the range of 1% to 100% in camel milk powder. The commercial camel milk powder was used to verify the practicability of this method. The real-time PCR normalization system has a good exponential correlation (R² = 0.9822 and 0.9923) between ovine or bovine content and Ct ratio (specific/internal reference gene) and allows for the quantitative determination of ovine or bovine milk contents in adulterated camel milk powder samples. Accuracy was effectively validated using simulated adulterated samples, with recoveries ranging from 80% to 110% with a coefficient of variation of less than 7%, exhibiting sufficient parameters of trueness. The ordinary PCR qualitative detection and real-time PCR quantitative detection method established in this study proved to be a specific, sensitive, and effective technology, which is expected to be used for market detection.     

流动型微流控PCR扩增芯片的研究

组装了由注射泵进样系统、微流控芯片和三温区加热器组成的流动型PCR扩增系统,该系统具有扩增速度快、交叉污染小、芯片可重复使用和操作方便等特点.优化了芯片厚度、隔热材料和流速等影响PCR扩增的因素.在4.9min内经24个循环成功地扩增了浓度为1ng/100μL的λ-DNA(500bp).     

DNA Extraction from Formalin-fixed and Paraffin-embedded Tissues by Triton X-100 for Effective Amplification of EGFR Gene by Polymerase Chain Reaction

For first-line non-small-cell lung cancer(NSCLC) therapy, detecting mutation status of the epidermal growth factor receptor(EGFR) geneconstitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor(TKI) therapy. Now, the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded(FFPE) tissues. DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction(PCR) are two key steps for detecting EGFR gene mutation. We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene. Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100(pH=10.0) was performed by heating at 95℃ for 30 min. Meanwhile, a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison. DNA extracted products were used as template for amplifying the exons 18, 19, 20 and 21 of EGFR by PCR for different amplified fragments. Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250-500 base pairs(bp). However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp. The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit. For about 500 bp fragment, four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit. However, for about 200 bp fragment. This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR, further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.     

Real-time PCR for quantitative detection of chamois (Rupicapra rupicapra) and pyrenean ibex (Capra pyrenaica) in meat mixtures

A real-time quantitative polymerase chain reaction (PCR) technique was developed for the quantification of chamois and pyrenean ibex DNAs in meat mixtures by using a SYBR green detection platform. Two species-specific systems and a eukaryotic endogenous system were combined in the real-time PCR approach to quantify the target species. In the specific systems, a 133 base pair (bp) fragment of the 12S rRNA gene was amplified from chamois DNA, and an 88 bp fragment from the D-loop region was amplified from pyrenean ibex DNA. In the endogenous system, universal primers amplified a 141 bp fragment on the nuclear 18S rRNA gene from eukaryotic DNA. The threshold cycle values obtained with the 18S rRNA primers were used to normalize those obtained from chamois- or pyrenean ibex-specific systems, serving as endogenous control for the total content of PCR-amplifiable DNA in the sample. Analysis of experimental raw and heat-treated binary mixtures of chamois and pyrenean ibex meat in a swine meat matrix demonstrated the suitability of the assay for the detection and quantification of the target DNAs in the range of 0.1-0.8%, depending on the species and treatment of the meat samples.     

An effective sequence characterized amplified region‐PCR method derived from restriction site‐amplified polymorphism for the identification of female Schistosoma japonicum of zoonotic significance

In the present study, restriction site‐amplified polymorphism (RSAP) markers were used to examine the genetic variability of Schistosoma japonicum isolates from different endemic provinces in mainland China. Of the 45 pairs of primers screened, 10 RSAP markers showed a clear banding pattern with good resolution; however, only six exhibited a polymorphism among different isolates. Among six RSAP markers, one pair of primers (R8+R10) was able to differentiate male and female parasites, and amplified one constant specific band for female S. japonicum isolates. The specific band was recovered, re‐amplified and sequenced, and a sequence of 162 bp was obtained. Based on this sequence, a pair of specific primers was designed and used to develop sequence characterized amplified region (SCAR)‐PCR assay for identification and differentiation of female S. japonicum isolates. The SCAR‐PCR assay allowed the specific identification of female S. japonicum, with no amplicons being amplified from male S. japonicum, Fasciola hepatica, Clonorchis sinensis, S. mansoni (male and female parasite). DNA sequencing confirmed the identity of the amplified products. The minimum amount of DNA detectable using SCAR‐PCR assay was 0.3 ng for female S. japonicum. The SCAR‐PCR was able to differentiate effectively the male and female S. japonicum worms collected from 12 geographical origins in eight endemic provinces, the gender of which was known based on the morphological and biological features. These results showed that SCAR‐PCR provides an effective tool for the sex differentiation studies of S. japonicum, identification of female S. japonicum, diagnosis and epidemiological survey of S. japonicum infections in animals and human.     

Advantages of Thermococcus kodakaraenis (KOD) DNA Polymerase for PCR-mass spectrometry based analyses

The advantages of the thermostable DNA polymerase from Thermococcus kodakaraensis (KOD) are demonstrated for PCR amplification with subsequent detection by mass spectrometry. Commonly used DNA polymerases for PCR amplification include those from Thermus aquaticus (Taq) and Pyrococcus furiosus (Pfu). A 116 base-pair PCR product derived from a vWA locus was amplified by Taq, Pfu, or KOD DNA polymerase and compared by agarose gel electrophoresis and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). KOD DNA polymerase demonstrated a 2- to 3-fold increase in PCR product formation compared to Pfu or Taq, respectively, and generated blunt-ended PCR product which allows facile interpretation of the mass spectrum. Additionally, we demonstrate the advantage of using high magnetic fields to obtain unit resolution of the same 116 base pair (approximately 72 kDa) PCR product at high m/z.     

Real-time polymerase chain reaction detection of fishmeal in feedstuffs

A SYBR Green PCR system was developed for detection of fishmeal in feedstuffs. The real-time PCR method combines the use of fish-specific primers that amplify an 87 base pair (bp) fragment of the mitochondrial 12S ribosomal RNA gene from fish species, and a positive control primer pair that amplifies a 99 bp fragment of the nuclear 18S ribosomal RNA gene in all eukaryotic organisms. The specificity of the primers was tested against 52 animal species and six plant species. Reference feedstuff samples were successfully tested for the presence of fishmeal, demonstrating the applicability of the assay to feedstuffs.     

Application of Single—labelled Probe—primer in PCR Amplification to the Detection of Hepatitis B Virus DNA

A new method based on the incorporation of a single-lablled probe-primer into polymerase chain reaction(PCR) for the detection of PCR-amplified DNA in a closed system is reported.The probeprimerc consists of a specific probe sequence on the 5‘‘‘‘‘‘‘‘-end and a primer sequence on the 3‘‘‘‘‘‘‘‘-end.A flurophore is located at the 5‘‘‘‘‘‘‘‘end.The primeR-quencher is an oligonucleotide,which is complementary to the probe sequence of probe-primer and labelled with a quencher at the 3‘‘‘‘‘‘‘‘-end.In the duplex formed by probe-primer and primer-quencher.the fluorophore and quencher are kept in close proximity to each other.Therefore the fluorescence is quenched.During PCR amplificatio,the specific probe sequence of probeprimer binds to its complement within the same strand of DNA,and is cleaved by Taq DNA polymerase,resulting in the restoration of fluorescence.This system has the same energy transfer mechanism as molecular beacons,and a good quenching effciency can be ensured.Following optimization of PCR conditions,this method was used to detect hepatitis b virus(HBV) dna in patient sera.This technology eliminates the risk of carry-over contamination,simplifies the amplification assay and opens up new possibilities for the real-time detection of the amplified DNA.     

Flanking Sequence Determination and Event Specific Detection of Transgenic Wheat B72-8-11b Strain

Exogenous fragment sequence and flanking sequence between exogenous fragment and recombinant chromosome of transgenic wheat B72-8-11b were successfully acquired through PCR amplification with cross-matched primers from exogenous genes. Newly acquired exogenous fragment covered the full-length sequence of transformed genes such as transformed plasmid and corresponding functional genes including marker uidA, promoter ubiquitin, lacZ, 1Dx5, and part of sequence of the wheat genome. A specific PCR detection method for transgenic wheat B72-8-11b strain was established on the basis of primers designed according to flanking sequence. The designed primers revealed specific amplification of 132 bp product of transgenic wheat B72-8-11b strain. This method is characteristics of high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B72-8-11b strain.     

Developments toward a complete micro-total analysis system for Duchenne muscular dystrophy diagnosis

The diagnosis of Duchenne muscular dystrophy (DMD) has historically utilized either PCR or requires Southern blot analysis, a southern blot analysis, however, is not amenable to incorporation in a microdevice format. A PCR amplification-based method has been developed, and we have previously coupled this amplification with microchip separation of the PCR fragments for DMD diagnosis. Diagnoses of affected patients were performed by comparing exon concentrations to those of control samples amplified at the same time. To accurately identify mutations in patient samples, this work established normal ranges for the concentration of each amplified exon fragment using control samples amplified over successive days. Our studies show that the number of cycles used in the amplification process affects this range. Affected patient samples were analyzed using these normal ranges and the mutations detected by Southern blot analysis were also diagnosed using the microchip separation method.

Employing the microchip separation method decreases the time required for the analysis, but the time required for DNA purification and PCR amplification must also be decreased for faster total analysis of patient samples. Development of microchip methods for these processing steps is one approach for reducing the individual times, while also providing the possibility of integrating these steps in a single device. Here we report on the microchip extraction of genomic DNA from whole blood using a novel sol–gel matrix that is easily formed in microdevices. IR-mediated PCR amplification of a β-globin fragment from genomic DNA followed by electrophoretic analysis on a single integrated microdevice is presented for the first time. Work towards the development of a micro-total analysis device for DMD diagnosis, through integration of all processing steps on a single device, is also discussed.     

Electrophoretic detection of genetic variability among Schistosoma japonicum isolates by sequence-related amplified polymorphism

In the present study, sequence‐related amplification polymorphism (SRAP) was utilized to study the genetic variability among Schistosoma japonicum isolates from different provinces in China, using Schistosoma mansoni from Puerto Rico for comparison. Five out of ten tested SRAP primer combinations displayed significant polymorphisms among S. japonicum isolates from China, namely ME2/EM1, ME4/EM1, ME4/EM6, ME5/EM4 and ME5/EM5. Analysis of the 61 S. japonicum samples from China with five SRAP primer combinations identified a total of 83 reproducible polymorphic fragments. The number of fragments using each primer combination ranged from 14 to 19, with an average of 16 polymorphic bands per primer pair, and the size of fragment ranged approximately from 100 to 1000 bp. Representative‐specific SRAP fragments were excised from the gels, and confirmed by PCR amplification of genomic DNA using primers designed and based on the sequences of these SRAP fragments. Based on SRAP profiles, unweighted pair‐group method with arithmetic averages (UPGMA) dendrogram was constructed. UPGMA clustering algorithm categorized S. japonicum isolates from China into nine clades and two lineages (representing the mountainous and lake/marshland regions). These results indicate the usefulness of the SRAP technique for revealing genetic variability among S. japonicum isolates from China, and the SRAP technique should be applicable to other living organisms.     

A Genosensor for Point Mutation Detection of P53 Gene PCR Product Using Magnetic Particles

The development of an electrochemical genosensor involving DNA biotinylated capture probe immobilized on streptavidin coated paramagnetic beads and microfluidic based platform for the detection of P53 gene PCR product is reported. The novelty of this work is the combination of a sensitive electrochemical platform and a proper microfluidic system with a simple and effective enzyme signal amplification technology, ELISA, for detection of target DNA sequence and single nucleotide mutation in p53 tumor suppressor gene sequence. The biosensor has been applied to detect the PCR amplified samples and the results shows that it can discriminate successfully perfect matched DNA from mutant form.     

Effect of sustained elevated temperature prior to amplification on template copy number estimation using digital polymerase chain reaction

Digital polymerase chain reaction (dPCR) has the potential to enable accurate quantification of target DNA copy number provided that all target DNA molecules are successfully amplified. Following duplex dPCR analysis from a linear DNA target sequence that contains single copies of two independent template sequences, we have observed that amplification of both templates in a single partition does not always occur. To investigate this finding, we heated the target DNA solution to 95 °C for increasing time intervals and then immediately chilled on ice prior to preparing the dPCR mix. We observed an exponential decline in estimated copy number (R(2)≥ 0.98) of the two template sequences when amplified from either a linearized plasmid or a 388 base pair (bp) amplicon containing the same two template sequences. The distribution of amplifiable templates and the final concentration (copies per μL) were both affected by heat treatment of the samples at 95 °C from 0 s to 30 min. The proportion of target sequences from which only one of the two templates was amplified in a single partition (either 1507 or hmg only) increased over time, while the proportion of target sequences where both templates were amplified (1507 and hmg) in each individual partition declined rapidly from 94% to 52% (plasmid) and 88% to 31% (388 bp amplicon) suggesting an increase in number of targets from which both templates no longer amplify. A 10 min incubation at 95 °C reduced the initial amplifiable template concentration of the plasmid and the 388 bp amplicon by 59% and 91%, respectively. To determine if a similar decrease in amplifiable target occurs during the default pre-activation step of typical PCR amplification protocol, we used mastermixes with a 20 s or 10 min hot-start. The choice of mastermix and consequent pre-activation time did not affect the estimated plasmid concentration. Therefore, we conclude that prolonged exposure of this DNA template to elevated temperatures could lead to significant bias in dPCR measurements. However, care must be taken when designing PCR and non-PCR based experiments by reducing exposure of the DNA template to sustained elevated temperatures in order to improve accuracy in copy number estimation and concentration determination.