Real-time quantitative polymerase chain reaction (qPCR)

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Quantitative polymerase chain reaction (qPCR) identifies and quantifies microorganisms in environmental samples by detecting a unique target sequence of nucleic acid (DNA or RNA).

Photo of fluorescence amplification curves from a quantitative polymerase chain reaction (qPCR) analysis

Example fluorescence amplification curves from a quantitative polymerase chain reaction (qPCR) analysis.

To perform quantitative polymerase chain reaction (qPCR) analysis, samples are collected, concentrated, and the DNA or RNA is extracted. Cycles of heating and cooling are used to separate and copy complementary DNA strands containing the target sequence. Reaction components bind to the target sequence and allow the polymerase enzyme to create two copies from each original (one from each of the complementary strands). The heating/cooling cycle is repeated, and the target sequence is doubled each time. A fluorescent marker that is built in to each copy indicates the quantity of the target sequence, and the concentration is determined using a standard curve of known sequence concentrations.

LIDE performs qPCR for over 30 pathogens and fecal indicators, completing nearly 20,000 analyses per year. Additional qPCR assays are evaluated and optimized as needed. LIDE uses two Roche LightCycler* 480 machines, and two robotic nucleic acid extractors provide high sample throughput.

List of assays currently available for quantitative polymerase chain reaction (qPCR) analysis at LIDE:

BACTERIA

  • Bovine Bacteroides
  • Campylobacter
  • Clostridium
  • Cow M2 and M3 Bacteroides-like
  • Enterococcus
  • Enterohemorrhagic E. coli
  • Human Bacteroides
  • Human M2 Bacteroides-like
  • Mycobacterium avium subsp. paratuberculosis
  • Salmonella
  • Staphylococcus
  • Streptococcus

HUMAN VIRUSES

  • Hepatitis A virus
  • Human adenovirus
  • Human enterovirus
  • Human polyomavirus
  • Human rotavirus A
  • Norovirus (GI & GII)

BOVINE VIRUSES

  • Bovine adenovirus
  • Bovine coronavirus
  • Bovine enterovirus
  • Bovine polyomavirus
  • Bovine rotavirus (group A & C)
  • BVDV (Type 1 & 2)

OTHER VIRUSES

  • Avian influenza
  • Hepatitis E virus
  • Pepper mild mottle virus

PROTAZOA

  • Cryptosporidium
  • Giardia lamblia

ANTIBIOTIC RESISTANCE GENES

  • Tetracycline (A, W, X)
  • Erythromycin
  • Sulfanamide
  • Class 1 integrons

 

* Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.