[Discussion on Technical Evaluation Requirements of Allergen Detection Reagent Pre-marketing].

In the perspective of technical evaluation, the pre-marketing regulatory requirements of allergen detection reagents in China, America, European Union were compared, and the regulatory risks and performance requirements of this product were analyzed based on the monitoring of post-marketing adverse events, reference standards and domestic and foreign regulatory documents. In view of the "neck-stuck" problems such as the difficulty of clinical trials, the difficulty of finding comparable contrast reagents and the lack of clinical diagnostic gold standards, this paper discusses and gives regulatory suggestions, with a view to providing technical reference for product R&D, production, evaluation, approval and supervision in this field.

[Analysis of Class II Common Problems in the Registration of in Vitro Diagnostic Reagent (Kit) for Clinical Chemistry].

From the perspective of technical review, this paper made statistics on the supplement contents of in vitro diagnostic reagent (kit) for clinical chemistry registered in Zhejiang province in the past five years, summarized and analyzed the common problems, and put forward corresponding suggestions based on the common problems encountered in the public welfare training of registered specialists in Zhejiang province. The aim is to provide technical reference for registrars to prepare registration documents reasonably and efficiently and for review staffs to strengthen their points of focus.

ORF4-protein deficient PCV2 mutants enhance virus-induced apoptosis and show differential expression of mRNAs in vitro.

Porcine circovirus type 2 (PCV2) is the essential infectious agent of PCV associated disease (PCVAD). During previous in vitro studies, 11 RNAs and four viral proteins have been detected in PCV2-infected cells. Open reading frame (ORF) 4 is 180bp in length and has been identified at the transcription and the translation level. It overlaps completely with ORF3, which has a role in virus-induced apoptosis. In this study, start codon mutations (M1-PCV2) or in-frame termination mutations (M2-PCV2) were utilized to construct two ORF4-protein deficient viruses aiming to investigate its role in viral infection. The abilities of M1-PCV2 and M2-PCV2 to replicate, transcribe, express viral proteins, and to cause cellular apoptosis were evaluated. Viral DNA replication curves supported that the ORF4 protein is not essential for viral replication, but inhibits viral replication in the early stage of infection. Comparison of the expression level of ORF3 mRNA among wild-type and ORF4-deficient viruses in infected PK-15 cell demonstrated enhanced ORF3 transcription of both ORF4 mutants suggesting that the ORF4 protein may play an important role by restricting ORF3 transcription thereby preventing virus-induced apoptosis. This is further confirmed by the significantly higher caspase 3 and 8 activities in M1-PCV2 and M2-PCV2 compared to wild-type PCV2. Furthermore, the role of ORF4 in cell apoptosis and a possible interaction with the ORF1 associated Rep protein could perhaps explain the rapid viral growth in the early stage of infection and the higher expression level of ORF1 mRNA in ORF4 protein deficient PCV2 mutants.

Identification and characterization of two novel transcription units of porcine circovirus 2.

Porcine circovirus type 2 (PCV2) infection is associated with porcine circovirus-associated diseases in pigs, which is a serious threat to the swine industry worldwide. To date, only three open reading frames (ORFs) within the PCV2 genome have been reported: ORF1 codes for two replicase proteins (Rep and Rep'), ORF2 for the structural protein (Cap), and ORF3 for a protein implicated in cellular apoptosis. In this study, based on transcription analysis of ORF3 mRNA, a potential ORF4 mRNA was detected and characterized by real-time RT-PCR and rapid amplification of cDNA ends analysis. The results indicate that the ORF4 gene is expressed at the level of transcription in the PCV2-infected cells. In addition, a novel ORF3 associated (ORF3') mRNA was identified during virus replication in PK15 cells. Moreover, a 3' poly(A) addition signal sequence (AUUAAA, nt 258-263) was found 10-30 nucleotides upstream of the cleavage site in the novel ORF4 mRNA in the complementary-strand of the PCV2 genome. Furthermore, alternate trans-splicing was identified in the ORF3' mRNA between orientation diverse transcripts with typical GT-AG donor/acceptor junctions. Similar strategies as in this work can be applied to examine the transcription of other potential ORFs in PCV in the future.

Development of a single-tube multiplex real-time PCR for detection and identification of five pathogenic targets by using melting-curve analysis with EvaGreen.

SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye. Preferential binding of SG during PCR and inhibition of PCR often result in failure to detect multiple amplicons in multiplex reactions. In the present study, a novel single-tube, multiplex real-time PCR with EvaGreen dye (EG) was developed and evaluated for simultaneous detection of pathogenic targets by using five potato viruses as models. The PCR products obtained using five sets of specific primers were analyzed by melting curve analysis. The assay could specifically detect and differentiate the five potato viruses by producing a distinct peak for each amplification product and exhibited a high reproducibility with coefficients of variation from 0.01 to 0.25 %. Detection sensitivity of the assay ranged from 100 to 500 copies/µL for each virus. The results of this study demonstrate that multiplex real-time PCR and melting-curve analysis with EG is a sensitive, specific and inexpensive method for simultaneous detection of multiple pathogens.

A novel diagnostic platform based on multiplex ligase detection-PCR and microarray for simultaneous detection of swine viruses.

Simultaneous detection and identification of multiple pathogens is required in many diagnostic fields. In this study a novel method based on a multiplex ligase detection (LD)-polymerase chain reaction (PCR) and microarray (MLPM) is described to detect simultaneously several swine viruses involved in reproductive and/or respiratory problems. The multiplex diagnostic system was validated using standard plasmids, and clinical samples. Using this strategy as few as 10 copies of target plasmids were detected successfully. Each probe pair yielded specific positive signal only in its target site. In addition, when six target plasmids were present simultaneously sufficient robust signals were generated in their corresponding sites of six plasmid templates and no obvious signals were detected in non-target sites. Compared to real-time PCR, the MLPM showed specificities and sensitivities of 95.7-100% and 100% for 47 clinical samples tested, respectively. The results demonstrate that this novel assay is a specific, sensitive, and multiplex diagnostic method for detection of multiple pathogens and can also be adapted easily for diagnostic purposes.

Development of multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) mediated universal DNA microarray for diagnostic platform.

A multiplex reversal transcription-ligase detection reaction-polymerase chain reaction (MRLP) based universal microarray for the simultaneous pathogens detection was established by using potato viruses as a model. The proposed procedure integrated LDR for multiplicity and specificity, PCR amplification by universal primers for sensitivity, which required design of upstream and downstream LDR probes specific for each virus, and subsequent Zip-code microarray for multiplex and specific identification. Each MRLP fragments carried a unique sequence (complementary Zip-code sequence, cZip-code) which identified a virus by addressed to the location on the microarray where the Zip-code sequence has been spotted. Such Zip-code microarray and universal primers are therefore "universal" being unrelated to a specific molecular analyte. With this technique, target RNAs of ten potato viruses were reversal transcribed by random primer in a single reaction, then subjected to LDR and asymmetric labeling PCR as template, finally, the MRLP amplicons were analyzed by microarray hybridization and subsequent scanning. The technique platform was optimized and evaluated by using reference samples and artificial samples, which can specifically detect down to 3 copies of single or mixed plasmid templates. Due to its universality, multiplexing, specificity and sensitivity, this method can be recommended for simultaneously detecting a large number of different target types in related fields.

Reducing patulin contamination in apple juice by using inactive yeast.

The mycotoxin, patulin (4-hydroxy-4H-furo[3,2c]pyran-2[6H]-one), is a secondary metabolite produced mainly in rotten parts of fruits and vegetables, most notably apples and apple products, by a wide range of fungal species in the genera Penicillium, Aspergillus, and Byssochlamys. Due to its mutagenic and teratogenic nature and possible health risks to consumers, many countries have regulations to reduce levels of patulin in apple products. In the present study, reduction of patulin contamination in apple juice by using 10 different inactivated yeast strains was assessed. Our results indicated that nearly twofold differences in biomass existed among the 10 yeast strains. Eight of the 10 inactivated yeast strains could provide >50% patulin reduction in apple juice within 24 h, with the highest reduction rate being >72%. Furthermore, juice quality parameters, i.e., degrees Brix, total sugar, titratable acidity, color value, and clarity, of the treated apple juice were very similar to those of the untreated patulin-free juice. Potential applications of using inactivated yeast strain for patulin control are also discussed.