Specific detection of bovine coronavirus N protein with TaqMan probe qRT-PCR

Background: Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infectionmay cause losses to production by reduced weight gain and milk yield of the infected animals. Several methods have beenapplied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRTPCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technicalassay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene.Materials, Methods & Results: The present study was aimed to establish a novel TaqMan probe real-time PCR (qRT-PCR)for detecting bovine coronaviruses (BCoV), and also to develop a diagnostic protocol which simplifies sample collectionand processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from theknown sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimizedincluding concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, therecombinant plasmids of pMD18-T-BCoV-N were successfully constructed to make standard curves. The sensitivity, specificity and reproducibility were evaluated on the TaqMan qRT-PCR, respectively. A total of 321 feces specimens collectedfrom diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCRwere 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assaycould specially detect BCoV without detecting any other viruses. Its minimum detection limit was 4.72 × 101 copies/μL.However, universal PCR could detect only 4.72 × 103 copies/μL...(AU)

FSH receptor binding inhibitor up-regulates ARID1A and PTEN genes associated with ovarian cancers in mice.

Experiments were conducted to determine if the follicle-stimulating hormone (FSH) receptor binding inhibitor (FRBI) impacts the expression levels of AT-rich interactive domain-containing protein 1A (ARID1A) and phosphatase and tensin homolog (PTEN) in ovaries and blood, as well as expressions of follicle-stimulating hormone cognate receptor (FSHR) gene and proteins. Mice in FRBI-10, FRBI-20, FRBI-30, and FRBI-40 groups were intramuscularly injected with 10, 20, 30, and 40 mg FRBI/kg, respectively, for five consecutive days. Western blotting and qRT-PCR were utilized to determine expression levels of ARID1A and PTEN proteins and mRNAs. Serum ARID1A and PTEN concentrations of the FRBI-40 group were higher than the control group (CG) and FSH group (P<0.05). FSHR mRNA levels of FRBI-20, FRBI-30, and FRBI-40 groups were lower than that of CG and FSH groups on day 15 (P<0.05 or P<0.01). Expression levels of FSHR proteins of FRBI-30 and FRBI-40 groups were lower than those of CG and FSH groups (P<0.05). Levels of ARID1A and PTEN proteins of the FRBI-30 group were greater than CG on days 20 and 30 (P<0.05). FRBI doses had significant positive correlations to levels of ARID1A and PTEN proteins. Additionally, ARID1A and PTEN had negative correlations to FSHR mRNAs and proteins. A high dose of FRBI could promote the expression levels of ARID1A and PTEN proteins in ovarian tissues. FRBI increased serum concentrations of ARID1A and PTEN. However, FRBI depressed expression levels of FSHR mRNAs and proteins in mouse ovaries.

Synchronous detection of BPV and BVDV with duplex Taqman qPCR method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrheain dairy herds. BPV is a member of Bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirusgenus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes accordingto the 5UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viraldiarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the presentstudy was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV.Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed invitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, andthen used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV.Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probeqRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplificationconditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTRgene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. Forclinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealingtemperature was achieved in 43.2 for duplex BPV and BVDV. Dynamic curves and standard...(AU)

FSH receptor binding inhibitor up-regulates ARID1A and PTEN genes associated with ovarian cancers in mice

Experiments were conducted to determine if the follicle-stimulating hormone (FSH) receptor binding inhibitor (FRBI) impacts the expression levels of AT-rich interactive domain-containing protein 1A (ARID1A) and phosphatase and tensin homolog (PTEN) in ovaries and blood, as well as expressions of follicle-stimulating hormone cognate receptor (FSHR) gene and proteins. Mice in FRBI-10, FRBI-20, FRBI-30, and FRBI-40 groups were intramuscularly injected with 10, 20, 30, and 40 mg FRBI/kg, respectively, for five consecutive days. Western blotting and qRT-PCR were utilized to determine expression levels of ARID1A and PTEN proteins and mRNAs. Serum ARID1A and PTEN concentrations of the FRBI-40 group were higher than the control group (CG) and FSH group (P<0.05). FSHR mRNA levels of FRBI-20, FRBI-30, and FRBI-40 groups were lower than that of CG and FSH groups on day 15 (P<0.05 or P<0.01). Expression levels of FSHR proteins of FRBI-30 and FRBI-40 groups were lower than those of CG and FSH groups (P<0.05). Levels of ARID1A and PTEN proteins of the FRBI-30 group were greater than CG on days 20 and 30 (P<0.05). FRBI doses had significant positive correlations to levels of ARID1A and PTEN proteins. Additionally, ARID1A and PTEN had negative correlations to FSHR mRNAs and proteins. A high dose of FRBI could promote the expression levels of ARID1A and PTEN proteins in ovarian tissues. FRBI increased serum concentrations of ARID1A and PTEN. However, FRBI depressed expression levels of FSHR mRNAs and proteins in mouse ovaries.

Synchronous detection of BPV and BVDV with duplex Taqman qPCR method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrheain dairy herds. BPV is a member of Bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirusgenus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes accordingto the 5’UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viraldiarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the presentstudy was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV.Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed invitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, andthen used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV.Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probeqRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplificationconditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTRgene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. Forclinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealingtemperature was achieved in 43.2 for duplex BPV and BVDV. Dynamic curves and standard...

Specific detection of bovine coronavirus N protein with TaqMan probe qRT-PCR

Background: Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infectionmay cause losses to production by reduced weight gain and milk yield of the infected animals. Several methods have beenapplied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRTPCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technicalassay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene.Materials, Methods & Results: The present study was aimed to establish a novel TaqMan probe real-time PCR (qRT-PCR)for detecting bovine coronaviruses (BCoV), and also to develop a diagnostic protocol which simplifies sample collectionand processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from theknown sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimizedincluding concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, therecombinant plasmids of pMD18-T-BCoV-N were successfully constructed to make standard curves. The sensitivity, specificity and reproducibility were evaluated on the TaqMan qRT-PCR, respectively. A total of 321 feces specimens collectedfrom diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCRwere 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assaycould specially detect BCoV without detecting any other viruses. Its minimum detection limit was 4.72 × 101 copies/μL.However, universal PCR could detect only 4.72 × 103 copies/μL...