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1.
Comp Med ; 55(5): 419-24, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16270897

ABSTRACT

Mycoplasma pulmonis induces persistent infections in laboratory mice and rats and can contaminate biological materials. We developed a fluorogenic nuclease polymerase chain reaction (fnPCR) assay to detect M. pulmonis specifically. Primer and probe sequences for the assay were targeted to 16S rRNA sequences specific to M. pulmonis. The assay consistently detected the equivalent of fewer than 10 copies of template DNA. When evaluated against a panel of 24 species of bacteria, the M. pulmonis assay detected only M. pulmonis isolates. Evaluation of 10-fold serial dilutions of cultured M. pulmonis showed that the M. pulmonis fnPCR assay and culture on Dutch agar had comparable sensitivity in detecting viable M. pulmonis organisms, whereas the mouse antibody production test displayed positive serologic results at dilutions higher than those in which viable organisms could be detected. Finally, the M. pulmonis fnPCR assay was able to detect M. pulmonis DNA in nasopharyngeal wash fluid and trachea, lung, and uterus tissue collected from mice naturally infected with M. pulmonis but did not detect the organism in similar samples collected from uninfected, negative control mice. The M. pulmonis fnPCR assay provides a high-throughput, PCR-based method to detect M. pulmonis in infected rodents and contaminated biological materials.


Subject(s)
Mycoplasma pulmonis/isolation & purification , Polymerase Chain Reaction/methods , Animals , Base Sequence , DNA Primers , Female , Fluorescent Dyes , Male , Mice , Mice, Inbred C3H , Mice, Inbred ICR , Mycoplasma pulmonis/genetics , RNA, Ribosomal, 16S/genetics , Sensitivity and Specificity
2.
Comp Med ; 54(3): 288-92, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15253275

ABSTRACT

Lactate dehydrogenase-elevating virus (LDEV) induces persistent infections in laboratory mice, alters in vivo physiology, and is a common contaminant of biological materials such as transplantable tumor cell lines. The fluorogenic nuclease reverse transcriptase polymerase chain reaction (fnRT-PCR) assay combines RT-PCR analysis with an internal fluorogenic hybridization probe, thereby eliminating post-PCR processing and potentially enhancing specificity. An fnRT-PCR assay specific for LDEV was therefore developed by targeting primer and probe sequences to a unique region of the LDEV nucleocapsid (VP1) gene. Using the LDEV fnRT-PCR assay, we detected only LDEV and did not detect other RNA viruses that are capable of naturally infecting rodents. Using this assay, we detected as little as 10 fg of LDEV RNA; the assay was 10-fold less sensitive when directly compared with the mouse bioassay (measurement of serum LD after inoculation), without the problematic false-positive serum LD enzyme elevations associated with the mouse bioassay. Using the fnRT-PCR assay, we also were able to detect viral RNA in numerous tissues and in feces collected from experimentally inoculated C3H/HeN mice, but we did not detect any viral RNA in similar samples collected from age- and strain-matched mock-infected mice. Finally, using the fnRT-PCR assay, we were able to detect LDEV RNA in biological samples that had previously been determined to be contaminated with LDEV by use of the mouse bioassay and an RT-PCR assay at another laboratory. In conclusion, the LDEV fnRT-PCR assay is a potentially high-throughput diagnostic assay for detection of LDEV in mice and contaminated biological materials.


Subject(s)
Lactate dehydrogenase-elevating virus/isolation & purification , Animals , Biological Assay , Cloning, Molecular , DNA Primers , L Cells , Lactate dehydrogenase-elevating virus/genetics , Mice , Reverse Transcriptase Polymerase Chain Reaction/methods
3.
Comp Med ; 53(2): 173-7, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12784851

ABSTRACT

Sendai virus may induce acute respiratory tract disease in laboratory mice and is a common contaminant of biological materials. Pneumonia virus of mice (PVM) also infects the respiratory tract and, like Sendai virus, may induce a persistent wasting disease syndrome in immunodeficient mice. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proven useful for detection of Sendai virus and PVM immunodeficient animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, fnRT-PCR assays specific for Sendai virus and PVM were developed by targeting primer andprobe sequences to unique regions of the Sendai virus nucleocapsid (NP) gene and the PVM attachment (G) gene, respectively. The Sendai virus and PVM fnRT-PCR assays detected only Sendai virusand PVM , respectively. Neither assay detected other viruses of the family Paramyxoviridae or other RNA viruses that naturally infect rodents. The fnRT-PCR assays detected as little as 10 fg of Sendai virus RNA and one picogram of PVM RNA, respectively, andthe Sendai virus fnRT-PCR assay had comparable sensitivity when directly compared with the mouse antibody production test. The fnRT-PCR assays were also able to detect viral RNA in respiratory tract tissues and cage swipe specimens collected from experimentally inoculated C.B-17 severe combined immunodeficient mice, but did not detect viral RNA in age- and strain-matched mock-infected mice. In conclusion, these fnRT-PCR assays offer potentially high-throughput diagnostic assays to detect Sendai virus and PVM in immunodeficient mice, and to detect Sendai virus in contaminated biological materials.


Subject(s)
Murine pneumonia virus/isolation & purification , Pneumovirus Infections/veterinary , Respirovirus Infections/veterinary , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Sendai virus/isolation & purification , Animals , Fluorescent Dyes , Immunocompromised Host , Mice , Mice, Inbred ICR , Mice, SCID , Murine pneumonia virus/genetics , Murine pneumonia virus/pathogenicity , Pneumovirus Infections/transmission , Pneumovirus Infections/virology , RNA, Viral/analysis , Respirovirus Infections/transmission , Respirovirus Infections/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , Sendai virus/genetics , Sendai virus/pathogenicity , Sensitivity and Specificity , Severe Combined Immunodeficiency/veterinary , Severe Combined Immunodeficiency/virology
4.
Comp Med ; 53(1): 65-9, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12625508

ABSTRACT

Lymphocytic choriomeningitis virus (LCMV) induces persistent infections in laboratory mice; is a known contaminant of biological materials, such as transplantable tumor cell lines; and is of great concern in animal facilities due to its zoonotic potential. Fluorogenic nuclease reverse transcriptase-polymerase chain reaction (fnRT-PCR) assays combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. An fnRT-PCR assay specific for LCMV was, therefore, developed by targeting primer and probe sequences to a unique region of the LCMV nucleocapsid (NP) gene. The LCMV fnRT-PCR assay detected only LCMV and did not detect other RNA viruses that naturally infect rodents. The fnRT-PCR assay detected as little as one picogram of LCMV RNA, but was 100-fold less sensitive when directly compared with the mouse antibody production test. The fnRT-PCR assay was also able to detect viral RNA in numerous tissues and in feces and cage swipe specimens collected from experimentally inoculated BALB/c mice, but did not detect any viral RNA in similar samples collected from age- and strain-matched mock-infected mice. In conclusion, the LCMV fnRT-PCR assay offers a potentially high-throughput diagnostic assay to detect LCMV in mice and contaminated biological materials.


Subject(s)
Lymphocytic choriomeningitis virus/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Base Sequence , Cricetinae , DNA Primers , Female , Fluorescent Dyes , Lymphocytic choriomeningitis virus/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Pregnancy , RNA, Viral/analysis , Sensitivity and Specificity
5.
Comp Med ; 52(2): 111-6, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12022389

ABSTRACT

Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proved useful for the detection of mouse hepatitis virus (MHV) and rat coronavirus (RCV) in acutely infected animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays combine RT-PCR with an internal fluorogenic hybridization probe, thereby eliminating post-PCR processing and potentially enhancing specificity. Consequently, a fluorogenic nuclease RT-PCR assay specific for rodent coronaviruses was developed. Primer and probe sequences were selected from the viral genome segment that encodes the membrane (M) protein that is highly conserved among rodent coronaviruses. Use of the fluorogenic nuclease RT-PCR detected all strains of MHV and RCV that were evaluated, but did not detect other RNA viruses that naturally infect rodents. Use of the assay detected as little as two femtograms of in vitro transcribed RNA generated from cloned amplicon, and when compared directly with mouse antibody production tests, had similar sensitivity at detecting MHV-A59 in infected cell culture lysates. Finally, use of the assay detected coronavirus RNA in tissues, cage swipes, and feces obtained from mice experimentally infected with MHV, and in tissues and cage swipes obtained from rats naturally infected with RCV. These results indicate that the fluorogenic nuclease RT-PCR assay should provide a potentially high-throughput, PCR-based method to detect rodent coronaviruses in infected rodents and contaminated biological materials.


Subject(s)
Animals, Laboratory/virology , Coronavirus Infections/veterinary , Coronavirus, Rat/isolation & purification , Murine hepatitis virus/isolation & purification , Polymerase Chain Reaction/methods , Rodent Diseases/virology , Animals , Biological Assay/methods , Cell Line , Coronavirus Infections/virology , Coronavirus, Rat/genetics , Coronavirus, Rat/metabolism , Fluorescent Dyes/metabolism , Mice , Murine hepatitis virus/genetics , Murine hepatitis virus/metabolism , Rats , Rats, Sprague-Dawley , Sensitivity and Specificity
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