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CHILDREN'S HOSPITAL & RESEARCH CENTER

Molecular Diagnostics Tests Menu

The Clinical Molecular Diagnostics Laboratory at Children’s Hospital & Research Center Oakland uses a variety of molecular biology techniques for pathogen detection. The lab is a CLIA certified testing laboratory recognized by the College of American Pathologists (CAP) and the State of California as a licensed testing facility. Diagnostic molecular tests are only available to qualified professionals in the medical community.

CLIA Certificate#05D0643830
State of California Clinical Laboratory License#CLF261

For more information contact:
Kathleen Houtchens, PhD, MBADS
(510) 450-7629, FAX (510) 450-5692
Khoutchens@chori.org

For Genetic Testing, please our laboratory page.

Overview
Polymerase chain reaction (PCR) and related molecular biology techniques are rapid and sensitive methods for the detection of pathogens. Quantitation of viral load is also available using molecular techniques.


Test Menu*

Bordetella pertussis and parapertussis, Qualitative Detection by Real-Time PCR
Cytomegalovirus, Quantitative or Qualitative Detection by Real-Time PCR
Epstein-Barr virus, Quantitative or Qualitative Detection by Real-Time PCR
Herpes Simplex Virus 1 & 2, Qualitative Detection by Real-Time PCR
Respiratory Virus Panel by multiplex PCR

*Click on individual tests for descriptions, CPT codes and turnaround times.

Link to Requisition Form


Molecular Diagnostics Staff

Elizabeth Trachtenberg, PhD, Laboratory Director, Molecular Diagnostics
Margaret Vinson, CLS, General Supervisor, Molecular Diagnostics
Kathleen Houtchens, PhD, MBADS, Molecular Diagnostics
Calvin Ball, CLS
Susan Corkhill, CLS
Tricia Iacovangelo, CLS
Steve Morin, CLS
Jon Rowland, MD, PhD, Medical Director & Chair, Department of Pathology and Laboratory Medicine
Jayshree Merchant, Administrative Director, Department of Pathology and Laboratory Medicine


Bordetella pertussis and parapertussis, Qualitative Detection by Real Time PCR

Bordetella pertussis and parapertussis are closely related gram negative coccobacilli responsible for cyclical outbreaks of whooping cough occurring every three to five years. B. pertussis is a reportable disease.

Clinical Information[1]

  • A vaccine is available for pertussis but provides no significant protection against parapertussis
  • Children who are too young to be fully vaccinated are at risk for serious illness when infected with B. pertussis.
  • Infants under the age of 12 months have more serious illness from B. pertussis and are more likely to develop pneumonia, convulsions or other complications requiring hospitalization than persons in other age groups.
  • Infants are at greatest risk of fatality from B. pertussis.

Transmission

  • Bordetella is highly contagious; it is transmitted person to person via large respiratory droplets.

Methodology

  • Extraction of Viral nucleic acid using Qiagen EZ1 instrumentation and chemistry.
  • Real-time Polymerase chain reaction for the detection of target DNA using Eragen Analyte Specific Reagents on the Roche LightCycler 2.0.

Specimen requirements

  • Nasopharyngeal swabs with plastic or metal shafts in Universal Transport Media or equivalent are the only validated specimen.
  • Specimens should be obtained from the surface of ciliated epithelial cells.
  • Specimens from sputum, anterior nose or throat are not adequate [2].
  • Refrigerate specimen following collection, transport on ice.

Test Mnemonics

  • PERTPCR:                                    B. Pertussis/parapertussis PCR

CPT codes

  • 83890, 83900, 83901, 83912, 99001

Turnaround Time:

  • 72 hours

Detailed methodology

Nucleic acid is extracted using Qiagen’s EZ1 instrumentation and chemistry. Detection of the DNA target is via the Roche LightCycler 2.0 (LC 2.0) and Eragen Analyte Specific Reagents, a rapid and sensitive real-time PCR-based method for the detection of nucleic acids. B. pertussis primers target a 51 bp fragment in the multicopy insertion sequence IS481 (~240 copies/genome) and B. parapertussis primers target a 70 bp region in IS1001 (~20 copies/genome). Determination of the product-specific melting temperature (Tm) is carried out at the end of amplification, providing a second tier of specificity to the assay. A three degree shift in Tm is seen between B. pertussis and B. parapertussis in the analysis of Tm. A primer specific to the Internal Control (IC), controls for efficiency of the extraction as well as the PCR. The IC primers amplify a 72 bp fragment. Tm analysis is also carried out for the IC.

There are eight species in the genus Bordetella, some of these species are known to also harbor the insertion sequences IS481 and IS1001, the targets for the Eragen pertussis and parapertussis primers respectively (and always harbored by pertussis and parapertussis). For example Bordetella holmseii (ATCC strain 51541) harbors multiple copies of an IS481-like element and three to five copies of an IS1001-like element that shares only 87% sequence similarity [1]. This strain has been isolated from human nasopharyngeal specimens (NP) and has occasionally been associated with respiratory disease [3], [4]. The public health significance of the species remains to be determined; some reports suggest that it is only found in immunocompromised individuals[5]. In our laboratory the Eragen B. pertussis primers amplified a presumptive IS481 from Bordetella holmseii (ATCC strain 51541) but did not amplify any IS1001– thus we cannot rule out false positives for B. pertussis that would be generated in an individual infected with B. holmseii.

A B. bronchispetica strain derived from horses was shown to harbor one or two copies of IS481 (% similarity not analyzed) and B. bronchispetica strains derived from cats, dogs, mice and cattle harbor IS1001 (copy number and % similarity not noted[6].  However infection with B. bronchispetica is very rare in humans [7].

References

1. CDC Website
2. Mattoo, S. and J.D. Cherry, Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev, 2005. 18(2): p. 326-82.
3. Mazengia, E., et al., Recovery of Bordetella holmesii from patients with pertussis-like symptoms: use of pulsed-field gel electrophoresis to characterize circulating strains. J Clin Microbiol, 2000. 38(6): p. 2330-3.
4. Yih, W.K., et al., Bordetella holmesii-like organisms isolated from Massachusetts patients with pertussis-like symptoms. Emerg Infect Dis, 1999. 5(3): p. 441-3.
5. Loeffelholz, M.J., et al., Detection of Bordetella holmesii using Bordetella pertussis IS481 PCR assay. J Clin Microbiol, 2000. 38(1): p. 467.
6. van der Zee, A., et al., Molecular evolution and host adaptation of Bordetella spp.: phylogenetic analysis using multilocus enzyme electrophoresis and typing with three insertion sequences. J Bacteriol, 1997. 179(21): p. 6609-17.
7. Wernli, D., et al., Evaluation of eight cases of confirmed Bordetella bronchiseptica infection and colonization over a 15-year period. Clin Microbiol Infect, 2010.

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Cytomegalovirus, Quantitative or Qualitative Detection by Real Time PCR

Cytomegalovirus (CMV) is a member of the β-Herpesviruses, a family of double stranded DNA viruses in the Herpesviridae family. In developed countries, seroprevalence is approximately 50% in young adults. The primary sites of infection are monocytes and lymphocytes.  [1, 2]

Clinical Information [1, 2]

  • Generally asymptomatic in adults and immunocompetent children.
  • Can cause significant disease in neonates, immunocompromised and transplant patients.

Transmission

  • The virus is transmitted via bodily secretions including saliva, breast milk, vaginal fluids, semen, urine, and stool and remains dormant following primary infection.
  • Following primary infections, a latent infection is established.

Methodology

  • Extraction of Viral nucleic acid using Qiagen EZ1 instrumentation and chemistry
  • Real-time Polymerase chain reaction for the detection of target DNA using Roche Analyte Specific Reagents on the Roche LightCycler 2.0.

Specimen requirements

  • Plasma (ACD, EDTA), minimum volume: 1.0 ml.
  • Urine, minimum volume: 1.0 ml.
  • Bronchial Wash, minimum volume: 1.0 ml.
  • CSF, minimum volume: 0.5 ml.
  • Refrigerate specimen following collection, transport on ice.

Unacceptable Specimens

  • Whole Blood, Tissue, Serum, heparinized or hemolyzed plasma.

Test Mnemonics

  • CMVPBQL:                                    CMV PCR Plasma Qualitative
  • CMVPPQT:                                    CMV PCR Plasma Quantitative
  • CMVPBWQL:                                    CMV PCR Bronchial Wash Qualitative
  • CMVPBWQT:                                    CMV PCR Bronchial Wash Quantitative
  • CMVPCSFQL:                                    CMV PCR CSF Qualitative
  • CMVPCSFQT:                                    CMV PCR CSF Quantitative
  • CMVPUAQL:                                    CMV PCR Urine Qualitative
  • CMVPUAQT:                                    CMV PCR Urine Quantitative

CPT codes                                   

  • 83890, 87798 (QL), 87799 (QT), 83912, 99001

Turnaround Time

  • 72 hours

Detailed methodology

Viral nucleic acid is extracted using Qiagen’s EZ1 instrumentation and chemistry. Detection of the DNA target is via the Roche LightCycler 2.0 (LC 2.0) and Roche Analyte Specific Reagents, a rapid and sensitive real-time PCR- based method for the detection and quantification of nucleic acids.

The DNA target sequence is a 240-bp fragment of the CMV DNA polymerase gene UL54. The target is amplified with sequence specific primers for a highly conserved region of the gene. Presence of CMV amplicon is then detected with a pair of Fluorescence Resonance Energy Transfer (FRET) hybridization probes that anneal to an internal sequence of the amplified gene fragment. One of the probes is labeled with LC Red 640; the other probe is labeled with fluorescein. When the two probes come in proximity to each other energy is transferred from the donor fluorophore (fluorescein) to the acceptor Red 640 fluorophore, which then emits a signal that can be measured by the LC. The amount of signal is proportional to the amount of PCR product bound to probe.

A synthetic DNA molecule with the same binding sites, GC content and amplicon length (a cloned fragment of CMV UL54) serves as an Internal Control (IC) for both PCR and the extraction procedure. The IC is spiked into the specimens prior to extraction and detected with a unique IC hybridization probe labeled with the an acceptor Red 705 fluorophore (emitting light at 710 nm) which is detected in channel 3 of the LightCycler simultaneously with the detection of the LC Red 640 in a method known as “dual color detection” [3], [4].

References
1. CDC Website  
2. emedicine. http://emedicine.medscape.com/article/218580-overview.
3. Roche, Overview of LightCycler Quantitation Methods. Roche Applied Science
Technical Note No. LC 10/update 2003
4. Espy, M.J., et al., Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev, 2006. 19(1): p. 165-256.

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Epstein-Barr virus, Quantitative or Qualitative Detection by Real Time PCR

Epstein-Barr virus (EBV) or human Herpesvirus 4 is a Gamma-Herpes virus, a family of double stranded DNA viruses in the Herpesviridae family. In the US 50% of the population has been infected with EBV by age 5 and up to 90% by age 25. [1, 2]

Clinical Information [1, 2]

  • Primary infection is often asymptomatic.
  • Primary infection in adolescents and young adults may lead to infectious mononucleousis.
  • EBV is also associated with Post-Transplant Lymphoproliferative Disease (PTLD) and other cancers including Hodgkin’s lymphoma.

Transmission [1, 2]

  • The usual route of infection is salivary contact.
  • Following primary infections, a latent infection is established in lymphoid cells.

Methodology

  • Extraction of Viral nucleic acid using Qiagen EZ1 instrumentation and chemistry.
  • Real-time Polymerase chain reaction for the detection of target DNA using Roche Analyte Specific Reagents on the Roche LightCycler 2.0.

Specimen requirements

  • Whole blood (ACD, EDTA), minimum volume: 0.5 ml.
  • CSF, sterile, minimum volume: 0.5 ml.
  • Refrigerate specimen following collection, transport on ice.

Unacceptable Specimens

  • Tissue, Plasma, Serum, heparinized or hemolyzed whole blood.

Test Mnemonics

  • EBVPBQL:                                    EBV PCR Whole Blood Qualitative
  • EBVPBQT:                                    EBV PCR Whole Blood Quantitative
  • EBVPCSFQL:                                    EBV PCR CSF Qualitative
  • EBVPCSFQT:                                    EBV PCR CSF Quantitative

CPT codes                                    

  • 83890, 87798 (QL), 87799 (QT), 83912, 99001

Turnaround Time:

  • 72 hours

Detailed methodology [3, 4]

The DNA target sequence is a 239-bp fragment of the latent membrane protein 2 (lmp2) gene from EBV. The target is amplified with sequence specific primers for a highly conserved region of the gene. Presence of EBV amplicon is then detected with a pair of Fluorescence Resonance Energy Transfer (FRET) hybridization probes that anneal to an internal sequence of the amplified gene fragment. One of the probes is labeled with LC Red 640; the other probe is labeled with fluorescein. When the two probes come in proximity to each other energy is transferred from the donor fluorophore (fluorescein) to the acceptor Red 640 fluorophore, which then emits a signal that can be measured by the LC. The amount of signal is proportional to the amount of PCR product bound to probe.

A synthetic DNA molecule with the same binding sites, GC content and amplicon length (a cloned fragment of EBV lmp2 gene) serves as an Internal Control (IC) for both PCR and the extraction procedure. The IC is spiked into the specimens prior to extraction and detected with a unique IC hybridization probe labeled with the an acceptor Red 705 fluorophore (emitting light at 710 nm) which is detected in channel 3 of the LightCycler simultaneously with the detection of the LC Red 640 in a method known as “dual color detection” [4].

References

1. USCSM. Microbiology and Immunology On-line.  
2. CDC Website  
3. Fan, H. and M.L. Gulley, Epstein-Barr viral load measurement as a marker of EBV-related disease. Mol Diagn, 2001. 6(4): p. 279-89.
4. Roche, Overview of LightCycler Quantitation Methods. Roche Applied Science Technical Note No. LC 10/update 2003

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Herpes Simplex Virus 1 & 2, Qualitative Detection by Real Time PCR

Herpes Simplex Virus 1 & 2 (HSV 1& 2) are members of the α-Herpesviruses, a family of a double stranded DNA viruses in the Herpesviridae family renowned for their neurotropism. Seroprevalence for HSV-1 is 70- 80% in the U.S and 10-40% for HSV-2. There are numerous sites of infection including skin, lips, oral cavity, eyes, genital tract, and central nervous system (CNS). [1, 2]

Clinical Information [3,4,5]

  • Up to 80% of infections are asymptomatic.
  • Systemic involvement may occur.
  • HSV neonatal disease can lead to significant neurodevelopmental impairment or death.

Transmission

  • The virus is transmitted via bodily secretions onto susceptible mucosum or cracks in the skin.
  • Latency is established in neurons; viral shedding can occur during latent phase

Methodology

  • Extraction of Viral nucleic acid using Qiagen EZ1 instrumentation and chemistry.
  • Real-time Polymerase chain reaction for the detection of target DNA using Roche Analyte Specific Reagents on the Roche LightCycler 2.0.

Specimen requirements

  • Plasma (ACD, EDTA), minimum volume: 1.0 ml.
  • Swabs with plastic or metal shafts in Universal Transport Media (or equivalent).
  • CSF, minimum volume: 0.5 ml.
  • Refrigerate specimen following collection, transport on ice.

Unacceptable Specimens

  • Whole Blood, Tissue, Serum, heparinized or hemolyzed plasma.
  • Swabs with wooden shafts
  • Grossly hemolyzed CSF

Test Mnemonics

  • HSVDNA:                                    HSV PCR Plasma Qualitative
  • HSVPCSFQL:                                      HSV PCR CSF Qualitative
  • HSVPPQL:                                    HSV PCR Plasma Qualitative
  • HSVPSWQL:                                    HSV PCR Swab Qualitative

CPT codes:                                    

  • 83890,87798,83912,99001

Turnaround Time:

  • 72 hours

Detailed methodology

Viral nucleic acid is extracted using Qiagen’s EZ1 instrumentation and chemistry. Detection of the DNA target is via the Roche LightCycler 2.0 (LC 2.0) and Roche Analyte Specific Reagents, a rapid and sensitive real-time PCR- based method for the detection and quantification of nucleic acids.

The DNA target sequence is the HSV DNA polymerase gene. The target is amplified with sequence specific primers for a highly conserved region of the gene. Presence of HSV amplicon is then detected with a pair of Fluorescence Resonance Energy Transfer (FRET) hybridization probes that anneal to an internal sequence of the amplified gene fragment. One of the probes is labeled with LC Red 640; the other probe is labeled with fluorescein. When the two probes come in proximity to each other energy is transferred from the donor fluorophore (fluorescein) to the acceptor Red 640 fluorophore, which then emits a signal that can be measured by the LC. The amount of signal is proportional to the amount of PCR product bound to probe.

A synthetic DNA molecule with the same binding sites, GC content and amplicon length (a cloned fragment of HSV DNA pol) serves as an Internal Control (IC) for both PCR and the extraction procedure. The IC is spiked into the specimens prior to extraction and detected with a unique IC hybridization probe labeled with an acceptor Red 705 fluorophore (emitting light at 710 nm) which is detected in channel 3 of the LightCycler simultaneously with the detection of the LC Red 640 in a method known as “dual color detection” [4]

References

1.  CDC Website
2.  USCSM. Microbiology and Immunology On-line.  
3.  emedicine. http://emedicine.medscape.com/article/218580-overview.
4.  Espy, M.J., et al., Diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR. J Clin Microbiol, 2000. 38(2): p. 795-9.
5.  Whitley, R.J. and D.W. Kimberlin, Viral encephalitis. Pediatr Rev, 1999. 20(6): p. 192-8.

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Respiratory Virus Panel by multiplex PCR

Respiratory viruses are the most common cause of acute illness in the U.S. [1] Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza B, Respiratory Syncytial Virus subtype A, Respiratory Syncytial Virus subtype B, Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus, Human Metapneumovirus, Rhinovirus, and Adenovirus can be detected/differentiated using the Luminex xTAG Respiratory Virus Panel.

Clinical Information [2, 3]

  • The highest frequency of acute respiratory illness is found in children and adults over 55 years of age.
  • Respiratory viruses are implicated in many acute illnesses with the potential to cause serious disease in young, elderly and immunocompromised patients.
  • It is estimated that Influenza A, Influenza B, RSV A and B (RSV), Adenovirus, and Parainfluenza 1, 2, and 3 account for at least 80% of Lower Respiratory Tract infections (LRTI). [2]
  • In infants, LRTI can lead to serious complications including pneumonia and acute respiratory failure

Transmission

  • The viruses are transmitted via respiratory droplet. [2]

Methodology

  • Extraction of Viral nucleic acid using Qiagen EZ1 instrumentation and chemistry.
  • Multiplex Reverse-transcriptase Polymerase Chain Reaction (rt-PCR) followed by liquid phase microarrays using Luminex x-TAG reagents and a Luminex flow cytometry based platform.

Specimen requirements

  • Nasopharyngeal Swabs with plastic or metal shafts in Universal Transport Media (or equivalent) are the only validated specimen.
  • Refrigerate specimen following collection, transport on ice.

Unacceptable Specimens

  • Swabs with wooden shafts, or calcium alginate tips.

Test Mnemonics

  • RVP:                                                      Respiratory Virus Panel PCR

CPT codes                                   

  • 83890, 87798 (x 12), 83912, 99001

Turnaround Time:

  • 72 hours

Detailed methodology

Viral nucleic acid is extracted using Qiagen’s EZ1 instrumentation and chemistry. Detection of the nucleic acid target is via a bead based technology that allows for simultaneous detection of many targets using liquid phase micro arrays combined with flow cytometry signal detection. Multiplex reverse transcription- PCR (RT-PCR) of the target nucleic acid is followed by target specific primer extension and hybridization to an array of beads harboring target specific complementary sequences coupled to a unique fluorescent fingerprint. Excitation of the beads by laser allows for the identification of the unique fluorescent fingerprint and the presence or absence of the target specific product.

A recombinant RNA molecule from the MS2 bacteriophage coat protein gene acts as an Internal Control (IC) for both the reverse transcription and the extraction procedure. It is spiked into the specimens prior to extraction. Bacteriophage lambda DNA is used as a run control for the PCR and primer extension steps of the procedure.

References

1. Rotbart, H.A. and F.G. Hayden, Picornavirus infections: a primer for the practitioner. Arch Fam Med, 2000. 9(9): p. 913-20.
2. CDC Website 
3. emedicine. http://emedicine.medscape.com/article/218580-overview.

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revised: Thursday, June 16, 2016 11:50 AM

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