A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples

A sensitive and rapid single step real time （rt） RT-PCR was standardized using one-step Brilliant SYBR Green kit for detection and semi-quantitation ofpeste des petitis ruminants virus （PPRV） using the virus RNA and matrix （M） protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit （sensitivity） of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was -0.0001 cell culture infectious dose 50% units （TCID50）. Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection （dpi） and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.

Detection of Porcine Epidemic Diarrhea Virus in Guangxi Province from 2011 to 2014 and Sequence Analysis of Its M Gene

Detection of pigs epidemic diarrhea virus （PEDV） was conducted on 331 piglets diarrhea fecal samples collected in Nanning, Yulin and other 12 areas of Guangxi Province from January of 2011 to April of 2014 by the method of reverse transcription-polymerase chain reaction （RT-PCR）. The results showed that the positive samples of PEDV were 210 and the positive rate was 63.44%. The clone and sequencing of M gene was carried out on 25 positive samples. PEDV reference strains were selected from GeneBank to conduct the sequence homology alignment analysis and the phylogenetic tree of M gene. The M gene homology and amino acid sequence identity between 25 isolated strains and 51 reference strains were 96.0% - 99.6% and 94.3% - 99.6%, respectively. The genetic variation anal- ysis of M gene showed that the genetic relationship of PEDV prevalent strains in Guangxi Province from 2013 to 2014 was close to that of the prevalent strains in Bei- jing, Anhui, Wuhan, Hebei and Guangdong from 2010 to 2013, and which were far from that of the Chinese early isolates CH/S （GenBank number： JN547228 ）, vaccine strain CV777 （GenBank number： AF353511 ） and Attenuated DR13 （GenBank number： JQ023162）. Indicating that the PEDV strains prevalent in Guan- gxi in recent years showed significant variation with the early isolates.

Expression of MCMV M83 mRNA in Infected Testes and Its Relationship with Testicular Lesions

Objective To investigate the expression of routine cytomegalovirus （MCMV） M83 mRNA in mouse testes at the different periods of MCMV infection and its relationship with the severity of testicular lesions Methods Used digoxin-labeled MCMV mRNA oligonucleotide probe, the mouse testicular tissues at the different periods following MCMV inoculation were detected by in situ hybridization（ISH）. The average absorbency （A） of ISH positive signals represented the resticular MCMV load. The expression of MCMV M83 mRNA in the infected testes and its relation with the severity of testicular lesions were analyzed. Results At different periods after MCMV inoculation, the expression of MCMV M83 mRNA was detectable in the testicular tissues. The ISH positive signals were mainly located in cytoplasm of the testicular interstitial cells. The average A values at d 9 after MCMV infection were the highest （comparison between the experimental groups, P〈O. O01）. There were pathological inJTammatory changes to varying degrees in seminiferous epithelia and interstitial cells at the diJferent periods of infection, especially during d 6 to d 9. Conclusion In testicular MCMV infection, the levels of MCMV M83 mRNA were related with the severity of testicular lesions, suggesting the dynamic detection of MCMV M83 mRNA could evaluate the prognosis of MCMV infection.

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

H5N1 鸟的流行性感冒病毒(A/chicken/Hubei/489/2004 ) 的 M 和 NP 基因被 RT-PCR 从病毒的 RNA 放大，并且分别地克隆向量进 pMD18-T。包含 M 基因(pHM6-m ) 或 NP 基因(pHM6-np ) 的表示 plasmid 然后被把 M 或 NP 基因插入到 pHM6 优核质表示向量构造；构造 plasmid 然后被定序。32 只 BALB/c 老鼠(6-week-old ) 在随机被划分成四个组。三组 BALB/c 老鼠被接种一次有 plasmid pHM6-m， plasmid pHM6-np 的 30 渭 g 或 plasmid pHM6-m (15 渭 g ) 和 pHM6-np (15 渭 g ) 的混合的任何一个 30 渭 g 的肌内的线路分别地。老鼠的一个另外的组作为控制与 100 渭 l PBS 被注射。二个星期以后，所有老鼠与相应 H5N1 鸟的流行性感冒病毒被质问，并且在下列 12 天内观察了。在 pHM6-m 组， pHM6-np 组和混合 plasmids 组的老鼠的幸存率分别地是 62.5% ， 25.0% 和 50.0% 。结果证明有效保护能被 pHM6-m 或 pHM6-np 提供，但是 pHM6-m 比 pHM6-np 提供了更好保护的效果。关键词 H5N1 流行性感冒病毒 - M 基因 - NP 基因 - 克隆 - DNA 疫苗的 CLC 数字 S852.65 基础条款：国家基本科学才能训练资助(NSFC J0630648 )

Cloning and Expression Analysis of Mlo Gene from Pericallis hybrida B. Nord.

The full-length Mlo gene was obtained by reverse transcription polymerase chain reaction （RT-PCR） and RACE. The result of sequence analysis indicated that M/o gene from Pericallis hybrida B. Nord. contained about 1296bp open reading frame and encoded 431 amino acids. According to the comparison of the exogenous gene sequences by BLAST analysis and phylogenetic analysis, Mlo gene shared over 85% nucleotide homology and 98% amino acid homology. Finally, through semi-quantitative-PCR and fluorescence quantitative analysis, we found that Mlo gene showed the highest expression levels in leaves and the lowest in roots after inoculated with powdery mildew pathogen for different days.

All-trans retinoic acid as a single agent induces complete remission in a patient with acute leukemia of M_(2a) subtype

Objective To present a special case with the karyotype and molecular marker of acute myeloid leukemia (AML)-M 2 who was induced to complete remission by all-trans retinoic acid (ATRA) alone.Methods A recently hospitalized young female patient with acute leukemia was initially diagnosed as M 3 subtype based on morphological French-American-British (FAB) classification. Karyotype analysis using standard G and R banding techniques and RT-PCR were applied to further define the diagnosis. After primarily cultured bone marrow cells from the iliac aspiration were tested for in vitro induced differentiation, the patient was treated with oral all-trans retinoic acid alone, 60?mg per day until complete remission was achieved. Peripheral blood and bone marrow changes were monitored over the whole treatment course.Results The characteristic chromosomal aberration for M 3, the t(15;17) reciprocal translocation, was not found while a t(8;21) translocation was verified. Furthermore, an amplified product of the AML-1/ETO fusion gene instead of the PML/RARα fusion gene was detected by RT-PCR and the diagnosis was corrected from M 3 to M 2. Primary cultured bone marrow cells can be fully induced to terminal differentiation after 4 days exposure to ATRA. A hematological complete remission was achieved after 40 days treatment with ATRA as a single therapeutic agent, suggesting an alternative pathway mediating ATRA-induced myeloid differentiation. Conclusion A leukemia patient with a subtype other than M 3, such as M 2 in this case, may also be induced to complete remission by the mechanism of ATRA-induced terminal differentiation. This implies that there may be a pathway other than PML/RARα fusion gene product which mediates ATRA-induced myeloid maturation in leukemia cells.