Multiplex digital PCR: a superior technique to qPCR for the simultaneous detection of duck Tembusu virus, duck circovirus, and new duck reovirus.

Duck Tembusu virus (DTMUV), duck circovirus (DuCV), and new duck reovirus (NDRV) have seriously hindered the development of the poultry industry in China. To detect the three pathogens simultaneously, a multiplex digital PCR (dPCR) was developed and compared with multiplex qPCR in this study. The multiplex dPCR was able to specifically detect DTMUV, DuCV, and NDRV but not amplify Muscovy duck reovirus (MDRV), Muscovy duck parvovirus (MDPV), goose parvovirus (GPV), H4 avian influenza virus (H4 AIV), H6 avian influenza virus (H6 AIV), and Newcastle disease virus (NDV). The standard curves showed excellent linearity in multiplex dPCR and qPCR and were positively correlated. The sensitivity results showed that the lowest detection limit of multiplex dPCR was 1.3 copies/µL, which was 10 times higher than that of multiplex qPCR. The reproducibility results showed that the intra- and interassay coefficients of variation were 0.06-1.94%. A total of 173 clinical samples were tested to assess the usefulness of the method; the positive detection rates for DTMUV, DuCV, and NDRV were 18.5, 29.5, and 14.5%, respectively, which were approximately 4% higher than those of multiplex qPCR, and the kappa values for the clinical detection results of multiplex dPCR and qPCR were 0.85, 0.89, and 0.86, indicating that the two methods were in excellent agreement.

Development and application of a multiplex qPCR assay for the detection of duck circovirus, duck Tembusu virus, Muscovy duck reovirus, and new duck reovirus.

A multiplex qPCR assay was developed to simultaneously detect duck circovirus (DuCV), duck Tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), and novel duck reovirus (NDRV), but it did not amplify other viruses, including duck virus enteritis (DVE), infectious bursal disease virus (IBDV), avian reovirus (ARV), H5 avian influenza virus (H5 AIV), H7 avian influenza virus (H7 AIV), H9 avian influenza virus (H9 AIV), Newcastle disease virus (NDV), and Muscovy duck parvovirus (MDPV), and the detection limit for DuCV, DTMUV, MDRV, and NDRV was 1.51 × 101 copies/µL. The intra- and interassay coefficients of variation were less than 1.54% in the repeatability test with standard plasmid concentrations of 1.51 × 107, 1.51 × 105, and 1.51 × 103 copies/µL. The developed multiple qPCR assay was used to examine 404 clinical samples to verify its practicability. The positivity rates for DuCV, DTMUV, MDRV, and NDRV were 26.0%, 9.9%, 4.0%, and 4.7%, respectively, and the mixed infection rates for DuCV + DTMUV, DuCV + MDRV, DuCV + NDRV, MDRV + NDRV, DTMUV + MDRV, and DTMUV + NDRV were 2.7%, 1.2%, 1.2%, 1.0%, 0.5%, and 0.7%, respectively.

A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/µL (final reaction concentration of 12.1 copies/µL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses.

A Multiplex Crystal Digital PCR for Detection of African Swine Fever Virus, Classical Swine Fever Virus, and Porcine Reproductive and Respiratory Syndrome Virus.

African swine fever (ASF), classical swine fever (CSF), and porcine reproductive and respiratory syndrome (PRRS) are highly infectious diseases of domestic pigs and wild boars. The co-infections of ASF virus (ASFV), CSF virus (CSFV), and PRRS virus (PRRSV) have been reported in different pig farms. Early differential detection and diagnosis of ASFV, CSFV, and PRRSV in the clinical samples is very important for the effective prevention and control of these diseases. A multiplex crystal digital PCR (dPCR) was developed for differential detection of ASFV, CSFV, and PRRSV in this study, targeting p72, 5' untranslated region (UTR), and ORF7 genes, respectively. The different reaction conditions were optimized, and the specificity, sensitivity, and repeatability of the assay were evaluated. The results showed that the multiplex crystal dPCR was able to accurately and differentially detect ASFV, CSFV, and PRRSV with a limit of detection of 4.69 × 10-1 copies/µl, respectively, and could not detect other porcine viruses, i.e., foot-and-mouth disease virus (FMDV), Senecavirus A (SVA), atypical porcine pestivirus (APPV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV). The assay showed excellent repeatability and reproducibility, with coefficients of variation (CV) of the intra- and inter-assay from 0.09 to 1.40%, and from 0.64 to 2.26%, respectively. The 289 clinical samples from different pig herds in Guangxi province, China, were tested by the multiplex crystal dPCR and a reference multiplex real-time quantitative RT-PCR (qRT-PCR) established previously in our laboratory. The positive rates of ASFV, CSFV, and PRRSV were 30.10, 13.49, and 22.49% by the multiplex crystal dPCR, and 24.57, 8.65, and 18.34% by the multiplex qRT-PCR, with coincidence rates of 94.66, 95.16, and 95.84%, respectively. The results indicated that the established multiplex crystal dPCR was a specific, sensitive, and accurate method for the detection and quantification of ASFV, CSFV, and PRRSV. This is the first report on the multiplex dPCR for detecting ASFV, CSFV, and PRRSV.

Development of a one-step multiplex qRT-PCR assay for the detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus.

BACKGROUND: African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused great economic losses to the swine industry in China. Since coinfections of ASFV, CSFV and APPV occur in certain pig herds, it is necessary to accurately and differentially detect these pathogens in field-collected samples. In this study, a one-step multiplex real-time quantitative reverse transcription-polymerase chain reaction (multiplex qRT-PCR) was developed for the simultaneous and differential detection of ASFV, CSFV and APPV. RESULTS: The one-step multiplex qRT-PCR presented here was able to simultaneously detect ASFV, CSFV and APPV but could not amplify other viruses, including porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), porcine parvovirus (PPV), porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PRoV), porcine deltacoronavirus (PDCoV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV-1), BVDV-2, etc. The limit of detection (LOD) of the assay was 2.52 × 101 copies/µL for ASFV, CSFV and APPV. A repeatability test using standard recombinant plasmids showed that the intra- and interassay coefficients of variation (CVs) were less than 2%. An assay of 509 clinical samples collected in Guangxi Province, southern China, from October 2018 to December 2020 showed that the positive rates of ASFV, CSFV and APPV were 45.58, 12.57 and 3.54%, respectively, while the coinfection rates of ASFV and CSFV, ASFV and APPV, CSFV and APPV were 4.91, 1.38, 0.98%, respectively. Phylogenetic analysis based on the nucleotide sequences of the partial ASFV p72 gene showed that all ASFV strains from Guangxi Province belonged to genotypes I and II. CONCLUSION: A one-step multiplex qRT-PCR with high specificity, sensitivity and repeatability was successfully developed for the simultaneous and differential detection of ASFV, CSFV and APPV.

Development a multiplex RT-PCR assay for simultaneous detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus.

African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused considerable financial losses to the pig industry worldwide, and it is critical to achieve early and accurate diagnosis of these viruses to control the diseases induced by them. In this study, three pairs of specific primers were designed based on the highly conserved genome regions of these viruses, and a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay for ASFV, CSFV and APPV was established after various reaction conditions were optimized. The mRT-PCR assay consisted of two steps, that is, reverse transcription (RT) and mPCR. The assay was highly specific, sensitive, and reproducible for ASFV, CSFV and APPV without cross-reaction with other swine pathogens. The sensitivity of this assay, which used purified plasmid constructs containing specific viral target fragments as templates, was 6.34 × 102 copies/µL for ASFV and 6.34 × 101 copies/µL for both CSFV and APPV. A total of 384 clinical samples from piglets suspected to be infected in Guangxi Province, Southern China, during 2018-2019 were analyzed by the established mRT-PCR method. The results showed that the positive rates of ASFV, CSFV and APPV were 43.75 %, 13.28 % and 4.17 %, respectively, and the coinfection rates of ASFV/CSFV, ASFV/APPV and CSFV/APPV were 5.47 %, 1.83 % and 1.30 %, respectively. To understand the epidemiological characteristics of APPV, the newly discovered virus, in Guangxi Province, the clinical samples from APPV-positive animals were selected randomly for amplification and sequencing, and the complete genomic sequences of four APPV strains were obtained. Phylogenetic analysis demonstrated that APPV strains from Guangxi Province had a high degree of genetic diversity. This study provides an important tool for rapid detection and accurate diagnosis of ASFV, CSFV and APPV.

Evolution and genetic diversity of atypical porcine pestivirus (APPV) from piglets with congenital tremor in Guangxi Province, Southern China.

Atypical porcine pestivirus (APPV) was identified and associated with congenital tremor (CT) type A-II in new born piglets and has been reported in many countries. In China, the first APPV identification in swine herds was reported in Guangdong province in 2016. To investigate the genetic characteristics of APPV in Guangxi province, 53 tissue samples from neonatal piglets with CT were collected and detected from October 2017 to May 2019. Five APPV strains which were named as GX04/2017, GX01-2018, GX02-2018, GX01-2019 and GX02-2019 were obtained. Sequence analysis revealed that all six APPV strains from Guangxi province, including five strains from this study and one from a previous report, shared 83.3%-97.5% nucleotide identity of complete genome and 91.7%-99.1% amino acid identity of the open reading frame (ORF), and shared 77.7%-97.7% nucleotide identity of complete genome and 90.6%-99.3% amino acid identity of ORF with reference strains. Phylogenetic analysis indicated that all APPV strains could be divided into three clades based on the complete genome, Npro , Erns and E2 gene sequences, respectively; and the APPV strains from Guangxi province distributed in two clades (clades I and II). No sign of recombination was observed from Guangxi strains. Evolution analysis performed on the complete genome of 58 APPV strains showed that America, Europe and Asia strains during 2006-2019 evolved at a mean rate of 1.37 × 10-4 substitutions/site/year, and the most recent common ancestor (tMRCA) of them was estimated as 1,700.5 years ago. The findings of this study indicated that there existed a high degree of genetic diversity of APPV from Guangxi province, Southern China, which provided important information on the epidemiological features and evolutionary relationships of APPV.

[Sequencing and analysis of the complete genome of encephalomyocarditis virus strain GXLC isolated from swine].

The complete genome of encephalomyocarditis virus (EMCV)strain GXLC isolated from swine was sequenced and analyzed. Five overlapped gene fragments covering the entire open reading frame (ORF) were amplified by RT-PCR, and the 3'-untranslated region (UTR) and 5'-UTR were amplified by the 3'-rapid amplification of cDNA ends (RACE) and 5'-RACE method, respectively. The genome sequences of strain GXLC were obtained by assembling the sequences of RT-PCR-generated cDNA fragments. The length of the complete genome was 7 725 nucleotides (nt). The homology comparison and phylogenetic analysis of the nucleotide and deduced amino acid sequences between strain GXLC and other EMCV strains available in GenBank were performed. The results showed that the complete genome identity between GXLC strain and the strains from China, i.e. GX0601, GX0602, BJC3 and HB1 and the strains from other countries, i.e. CBNU, K3, K11, TEL-2887A, EMCV-R and PV21 was over 99%. The phylogenetic trees based on the complete genome, the structural protein or the non-structural protein gene sequences revealed that the tree topology was similar. All the EMCV strains could be divided into two groups: group I and group II, and group I could be subdivided into subgroup Ia and subgroup Ib. The strains from swine belonged to subgroup Ia or Ib, and the strains from mice belonged to subgroup Ia, while the strains from Sus scro fa belonged to group II. Strain GXLC, together with other EMCV isolates from China, belonged to subgroup Ia.

[The diagnostic value of flexible bronchoscopy in tracheobronchopathia osteochondroplastica].

OBJECTIVE: To enhance the knowledge of tracheobronchopathia osteochondroplastica (TO), and to describe the value of flexible bronchoscopic diagnosis and treatment for the disease. METHODS: The clinical data, bronchoscopic findings, histological results and the methods and effect of bronchoscopic treatment in 10 patients with TO admitted to Xiangya Hospital between June 2006 and July 2007 were retrospectively analyzed. RESULTS: There were 8 males and 2 females (mean age 46 +/- 16, range 33-76 years). The bronchoscopic appearance of TO was multiple whitish, hard nodules projecting into the tracheal lumen (mostly from the anterior and less from the lateral walls). The lesions were found most frequently in the trachea and major bronchi, and lobar and segmental bronchi were involved less frequently. Nodules were restricted to the anterolateral walls in 7 cases. The distribution of the lesions was diffuse in 5, confluent in 2 and scattered in 3 cases. Six patients received bronchoscopic management, including radiofrequency treatment for 2 patients and argon ion laser treatment for the other 4. The lesions in the airways were reduced and clinical symptoms improved to some extent after treatment. No severe complications occurred during and after the procedures. CONCLUSIONS: The diagnosis of TO can be easily underdiagnosed or misdiagnosed. Flexible bronchoscopy with histological examination is the main method for the diagnosis of TO. Radiofrequency and argon ion laser treatment are safe and effective.

[Bronchoscopic argon plasma coagulation therapy for bronchial carcinoma].

OBJECTIVE: To evaluate the effect of bronchoscopic argon plasma coagulation therapy on bronchial carcinoma. METHODS: Thirty-one bronchial carcinoma patients were diagnosed by bronchoscope and pathological tests, with or without atelectasis or obstructive pneumonia on chest X-ray or chest CT. Argon plasma coagulation therapy was performed through bronchoscope. The location of the airway lesions, the degree of obstruction, dyspnea index, and complications were evaluated. RESULTS: The patients with bronchial carcinoma were treated 1-4 times by bronchoscopic argon plasma coagulation therapy. Full effectiveness was achieved in 15 patients (48.4 %), partial in 12 (38.7 %), and mild in the other 4 (12.9 %). The overall effective rate was 100%. CONCLUSION: Bronchoscopic argon plasma coagulation therapy for bronchial carcinoma can remarkably reduce the tumor size, relieve clinical symptoms, and alleviate the obstruction caused by bronchial neoplasm. Bronchoscopic argon plasma coagulation therapy is an effective and safe method for patients with bronchial carcinoma.

[Analysis of bronchoscopy characteristics for 729 female patients with lung cancer.].

BACKGROUND: The aim of this article is to study features of the bronchoscopy signs in female lung cancer patients. METHODS: The bronchoscopy data of 729 female lung cancer patients enrolled between January 1994 and June 2007 was analyzed, retrospectively. RESULTS: Most of the patients were middle-aged female (57.0%), then were the elderly (28.5%), and the youth composed much lower (14.0%). The most common histopathology was adenocarcinoma (42.8%), followed by squamous cell carcinoma (23.9%) and small cell carcinoma (19.2%), and all of them increased in the past few years. The female lung cancers were more in the right lung (P <0.05), and the upper lobes (P <0.05). Among 729 female lung cancer patients, 92.0% had apparent signs. Most of adenocarcinoma had infiltrative changes (P <0.05), but most of squamous cell carcinoma and small cell carcinoma had proliferative changes (P <0.05). The most common sing of bronchoscopy in patients with atelectasis was proliferative changes (P <0.05), but the most common sings of bronchoscopy in patients with pleural effusion was infiltrative changes (P <0.05). CONCLUSIONS: This study suggests brochoscopy is an important approach in diagnosis of female lung cancer. Paying more attention to the lung cancer of female patients and examining with bronchoscopy would be helpful for earlier diagnosis.

[Medical thoracoscopy in the diagnosis of complicate pleural effusion].

OBJECTIVE: To evaluate the role of medical thoracoscopy in the diagnosis of the pleural effusion of unknown etiology. METHODS: The results of 36 patients with the pleural disease of unknown etiology diagnosed by medical thoracoscopy were retrospectively analyzed, including the pathologic results and the complications. RESULTS: Among the 36 patients, 35 were determined with positive rate of 97.2%, and no serious complications was found. CONCLUSION: Medical thoracoscopy is an important method of diagnosing complicate pleural effusion, and has high positive rate. It is a simple operation, with no serious complication, and fast recovery.