**1. Introduction**

A report published in the Lancet in 1998 described the case histories of 12 previously normal children who developed symptoms of autism or inflammatory bowel disease after having received the measles, mumps, and rubella (MMR) vaccine [1]. This paper formed the basis for Andrew Wakefield's subsequent claim to have identified a new type of gastrointestinal disease, termed autistic enterocolitis. Despite never explicitly asserting a link between the MMR vaccine and this supposedly new, regressive form of autism, the paper sparked a ma‐ jor health scare in the United Kingdom. It is probable that the uncertainty and controversy surrounding the relationship between measles and autism contributed to the fact that in 2004/05, about 1.9 million school children and 300,000 pre-school children were recorded as incompletely vaccinated against measles in England, including more than 800,000 children completely unvaccinated. Based on this, approximately 1.3 million children aged 2-17 years were susceptible to measles [2]. In 2006, a 13-year old boy, who had not received the MMR vaccine, became the first person in the UK for 14 years to die of measles and as a result of almost a decade of low MMR vaccination coverage across the UK, by 2008 the disease had once again become endemic.

In 2010 the Lancet fully retracted the 1998 publication from the public record, stating that it had "become clear that several elements of the 1998 paper by Wakefield *et al* are incorrect, contrary to the findings of an earlier investigation".The circumstances surrounding this pub‐ lication were subject to an extensive investigation and received a huge amount of publicity. Wakefield was found guilty of serious professional misconduct over the way he carried out his research and was struck off the medical register in 2010. A long statement released on 24 May 2010 includes the following key statements:

© 2013 Bustin; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**•** "In all the circumstances and taking into account the standard which might be expected of a doctor practising in the same field of medicine in similar circumstances in or around 1996-1998, the Panel concluded that Dr Wakefield's misconduct not only collectively amounts to serious professional misconduct, over a timeframe from 1996 to 1999, but also, when considered individually, constitutes multiple separate instances of serious profes‐ sional misconduct. Accordingly the Panel finds Dr Wakefield guilty of serious professio‐ nal misconduct" and

**•** "Accordingly the Panel has determined that Dr Wakefield's name should be erased from the medical register. The Panel concluded that it is the only sanction that is appropriate to protect patients and is in the wider public interest, including the maintenance of public trust and confidence in the profession and is proportionate to the serious and wide-rang‐

Why There Is no Link Between Measles Virus and Autism

http://dx.doi.org/10.5772/52844

83

There was far less publicity about the attempts to use molecular techniques to corroborate a link between measles virus (MeV) and "autistic enterocolitis". The major technique used was the fluorescence-based real-time polymerase chain reaction (qPCR), a ubiquitous techni‐

A key attraction of qPCR technology is its apparent simplicity: an assay consisting of combining oligonucleotides, PCR enzyme and buffer with a nucleic acid template to pro‐ duce a qPCR reaction is perceived as undemanding. This practical simplicity is comple‐ mented by the absence of any requirement for post-assay handling, as well as the development of user-friendly data analysis software that makes data generation and vis‐ ualisation in the shape of amplification plots remarkably simple. Indeed, qPCR is often described as a mature technology and as the "gold standard" for nucleic acid quantifica‐ tion. Whilst it is true that the technology is capable of exquisite sensitivity and specifici‐ ty, coupled with high reproducibility and accuracy, it is essential to understand that qPCR assays are made up of numerous, often divergent protocols that use different in‐

Whilst qPCR is the method of choice for the detection of DNA, the enzyme (*Taq* polymerase) used to copy the DNA template does not work well with RNA. Hence, if the amplification target is RNA, as it is for MeV, an enzymatic RNA-to-DNA conversion (reverse transcrip‐ tion, RT) step must be carried out before the DNA copying step can specifically amplify the target of interest. This variant of the qPCR is termed reverse-transcription (RT)-qPCR and the principle of this reaction is simple: RNA is reverse transcribed into single stranded DNA, either in a separate reaction or as a "one tube" assay that uses a different enzyme (*Tth* polymerase), as was the case with the experiments discussed below. There are several detec‐ tion chemistries, but in this case a very specific and widely used probe-based method was used (Figure 1B). Importantly, the use of fluorescent reporter molecules permits concurrent target amplification, detection and quantification as the assay proceeds [4]. Fluorescence is detected using dedicated qPCR instruments, which have (i) a controllable heating block that can hold a variety of temperatures and rapidly change between them, (ii) an excitation light source to excite the fluorochrome, (iii) a detector to register photon emissions and (iv) soft‐ ware that allows analysis of the data. Fluorescence emissions are collected from each sample tube and the levels of background fluorescence detected by the fluorimeter module of the qPCR instrument are established, providing a baseline that defines the sensitivity of the in‐

que used for the sensitive and specific detection of DNA (Figure 1).

struments, enzymes, buffers and non-identical targets.

ing findings made against him" [3].

**2. Technology and target**

**2.1.** *qPCR*

**Figure 1.** The polymerase chain reaction, a method for copying the same stretch of DNA several million-fold. **A.** A PCR reaction consists of double stranded DNA, two short DNA molecules ("primers") whose sequence is complementary to opposite strands of the DNA, a DNA synthesis enzyme ("*Taq* polymerase") and four nucleotide building blocks ("dNTPs"). The reaction mixture is heated to 95ºC to dissociate the sample DNA strands, then cooled to around 55ºC to allow the two primers to bind to their targets on the individual strands. Next, *Taq* polymerase makes two new strands of DNA at its optimal temperature of around 70ºC, using the original strands as templates, hence duplicating the original DNA. This procedure is repeated many times, leading to more than one billion exact copies of the original DNA segment. These can be detected by running the samples on a gel and staining with a DNA-binding dye. **B.** qPCR obviates the need for gel electrophoresis by using fluorescence to detect copied DNA. The qPCR method used for the detection of MeV uses a target-specific DNA molecule ("probe") that has a fluorescent dye at one end (R) and a quencher (Q) on the other. In the absence of target, the quencher prevents the dye from emitting light. In the pres‐ ence of target, the probe binds to its target and is degraded by the *Taq* polymerase. This separates the fluorescent label and the quencher and so results in the emission of light. Both the PCR and light detection are automated and detected in a single step by a dedicated instrument.

**•** "Accordingly the Panel has determined that Dr Wakefield's name should be erased from the medical register. The Panel concluded that it is the only sanction that is appropriate to protect patients and is in the wider public interest, including the maintenance of public trust and confidence in the profession and is proportionate to the serious and wide-rang‐ ing findings made against him" [3].

There was far less publicity about the attempts to use molecular techniques to corroborate a link between measles virus (MeV) and "autistic enterocolitis". The major technique used was the fluorescence-based real-time polymerase chain reaction (qPCR), a ubiquitous techni‐ que used for the sensitive and specific detection of DNA (Figure 1).
