Advances in Application of PCR Amplification in Etiologic Diagnosis

In recent years,with the rapid development of molecular biology diagnostic technology,many new polymerase chain reaction(PCR)technologies with high specificity and good sensitivity have gradually been developed.While expanding the range of detection methods,these technologies inevitably have some disadvantages.Therefore,in clinical pathogen diagnosis,medical personnel should choose the detection method according to the detection purpose and pathogen characteristics.In this paper,the basic principle,application scope,advantages and disadvantages and development of various emerging PCR diagnostic techniques are respectively described in order to provide a theoretical reference for the selection of pathogenic biological diagnostic techniques in the clinical practice.

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.

The EPSPS Pro106Ser substitution solely accounts for glyphosate resistance in a goosegrass(Eleusine indica) population from Tennessee, United States

Previous studies have documented the occurrence of glyphosate-resistant （GR） goosegrass （Eleusine indica （L.） Gaertn.） and, in at least some cases, resistance is due to an altered target site. Research was performed to determine if an altered target site was responsible for GR in a Tennessee, United States goosegrass population （TennGR）. DNA sequencing revealed a mutation in TennGR plants conferring the Prol06Ser 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） substitution previously identified in other GR populations. F2 populations were derived from TennGR plants crossed with plants from a glyphosate-susceptible population （TennGS） and analyzed for their response to glyphosate and genotyped at the EPSPS locus. Plants from the F2 populations segregated 1：2：1 sensitive：intermediate：resistant in response to a selec- tive dose of glyphosate, and these responses co-segregated with the EPSPS genotypes （PP106, PS106, and SS106）. To separately investigate the effect of the Prol06Ser substitution on GR, glyphosate dose-response curves and 50% effective dose （EDso） values were compared among the three genotypes and the two parental populations. The SS106 genotype was 3.4-fold resistant relative to the PP106 genotype, identical to the resistance level obtained when comparing the resistant and susceptible parental populations. We conclude that the mutation conferring a Prol06Ser EPSPS mutation is solely responsible for GR in the TennGR goosegrass population.

Cloning and Expression of the vp39 Gene of Bombyx mori Nuclear Polyhedrosis Virus in E.coli

The nuclear capsid protein gene (vp39) ofBombyx mori nuclear polyhedrosis virus (BmNPV) was amplified successfully by PCR technique and inserted into pGEM 3zf(+). The 5′ and 3′ terminal area of the amplified vp39 gene were sequenced with silver-staining dideoxy method. Bmvp39 gene was sub-cloned into the expression vector pRSET-A, and transformed intoE. coli BL21. This gene was highly expressed by IPTG induction. SDS-PAGE analysis showed that the expressed protein is about 38 kd, and the expressed amount reached maxium in 4 h with IPTG induction.

Sequencing of p35 Gene from Heliothis armigera Polyhedrosis Virus and Its Homologous Comparison

Using p35 gene primers of AcNPV, about 1 kb fragment was obtained by PCR from HaNPV DNA and was sequenced thereafter. It has a full reading frame encode 299 amino acids. Sharing identity of 94% in nucleotides and 84% in predict amino acids with AcNPV, a apoptosis inhibiting gene was found. According to comparison of p35 genes in five kinds of baculovirus, it is found that AcNPV, TnNPV and BmNPV share the most intimate blood relation. HaNPV is near the above three species. LsNPV is little remote from them. This result reconfirmed those we have done with gp37 genes and vp39 genes. It is more accurate to use conserved gene for species division than that of the serological identification.

Genomic DNA Extraction Method for Mulberry Varieties in Anhui Province

[Objective] The paper aimed to get a efficient, stable and low-priced DNA extraction method of mulberry leaves. [Method] With tender leaves of mulberry varieties in Anhui Province as materials, genomic DNA was extracted with CTAB method, and further identified by ultraviolet spectrophotometer, agarose gel electrophoresis and PCR methods. [ Result] The A260/A280 ratio of extracted DNA was 1.80 - 1.90, and the yield was 0. 187 -0.275μg/mg. With extracted DATA as the template, clear target bands were obtained by PCR. [Conclusion] Extracted DNA was good in quality, which could satisfy the needs of follow-up mo-lecular biological operation.

Recycling Isolation of Plant DNA,A Novel Method

DNA is one of the most basic and essential genetic materials in the field of molecular biology. To date, isolation of sufficient and good- quality DNA is still a challenge for many plant species, though various DNA extraction methods have been published. In the present paper, a recycling DNA extraction method was proposed. The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times, and correspondingly four DNA precipitations （termed as the 1st, 2nd, 3rd and 4th DNA sample, respectively） were conducted. This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method. This modified CTAB method was tested in eight plant species, wheat, sorghum, barley, corn, rice, Brachy- podium distachyon, Miscanthus sinensis and tung tree. The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species. The DNA samples were good templates for PCR amplification of both ISSR and SSR markers. The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods, and thus is an effective and universal DNA isolation method.

Ethnopharmacological Investigation and Rapid Authentication of Mongolian Patent Medicines Digeda

Objective To investigate Mongolian medicinal plants called Digeda and the prescriptions in Inner Mongolia region and to establish a molecular method for authentication of Digeda Mongolian patent medicines （MPMs）. Methods A field investigation was conducted on traditional uses of Digeda. After interviewed traditional healers in Mongolian, ethnopharmacological information of Digeda prescriptions was recorded in detail, including names, compositions, and traditional uses. And the total DNA from 10 MPMs has been amplified by three pairs of specific primers. Specific PCR products were further identified by sequence alignment with the known sequences already submitted in GenBank or own sequences. Results Fifteen Digeda plants and 29 Digeda prescriptions with their ethnopharmacological knowledge were collected. Ten MPM samples containing Lomatogonium rotatum, Viola philippica, and Corydalis bungeana were successfully evidenced by PCR with specific bands as raw materials. Conclusion Digeda should be further investigated in ethnopharmacology, which is a fundamental step toward developing efficacious natural drugs for various diseases. PCR amplification of specific allele is an easy and economical method, which can be used to identify highly processed MPMs and will assist in monitoring their qualities and legalities.